Bicyclic pyrrolyl amides as glycogen phosphorylase inhibitors

ABSTRACT

This invention relates to compounds of Formula Ior stereoisomers, pharmaceutically acceptable salts or prod rugs thereof or a pharmaceutically acceptable salts of the prodrugs. This invention also relates to pharmaceutical compositions comprising a compound of Formula I, and to methods of treatment of diabetes, insulin resistance, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, cataracts, hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, or tissue ischemia.

This application is a division of Ser. No. 10/117,370, filed Apr. 5,2002, now U.S. Pat. No. 6,576,653, which is a division of U.S. Ser. No.09/670,759, now U.S. Pat. No. 6,399,601, which claims benefit of U.S.Provisional Application Ser. No. 60/157,148, filed Sep. 30, 1999.

FIELD OF THE INVENTION

This invention relates to bicyclic pyrrolyl amides and pharmaceuticalcompositions comprising bicyclic pyrrolyl amides. This invention alsorelates to the treatment of diabetes, insulin resistance, diabeticneuropathy, diabetic nephropathy, diabetic retinopathy, cataracts,hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia,hyperlipidemia, atherosclerosis, and tissue ischemia, particularlymyocardial ischemia, using the bicyclic pyrrolyl amides.

BACKGROUND OF THE INVENTION

In spite of the early discovery of insulin and its subsequent widespreaduse in the treatment of diabetes, and the later discovery of and use ofsulfonylureas, biguanides and thiazolidenediones, such as troglitazone,rosiglitazone or pioglitazone, as oral hypoglycemic agents, thetreatment of diabetes remains less than satisfactory.

The use of insulin requires multiple daily doses, usually by selfinjection. Determination of the proper dosage of insulin requiresfrequent estimations of the sugar in urine or blood. The administrationof an excess dose of insulin causes hypoglycemia, with effects rangingfrom mild abnormalities in blood glucose to coma, or even death.Treatment of non-insulin dependent diabetes mellitus (Type II diabetes,NIDDM) usually consists of a combination of diet, exercise, oralhypoglycemic agents, e.g., thiazolidenediones, and in more severe cases,insulin. However, the clinically available hypoglycemic agents can haveside effects that limit their use, or an agent may not be effective witha particular patient. In the case of insulin dependent diabetes mellitus(Type I), insulin is usually the primary course of therapy. Hypoglycemicagents that have fewer side effects or succeed where others fail areneeded.

Atherosclerosis, a disease of the arteries, is recognized to be theleading cause of death in the United States and Western Europe. Thepathological sequence leading to atherosclerosis and occlusive heartdisease is well known. The earliest stage in this sequence is theformation of “fatty streaks” in the carotid, coronary and cerebralarteries and in the aorta. These lesions are yellow in color due to thepresence of lipid deposits found principally within smooth-muscle cellsand in macrophages of the intima layer of the arteries and aorta.Further, it is postulated that most of the cholesterol found within thefatty streaks, in turn, give rise to development of the “fibrousplaque,” which consists of accumulated intimal smooth muscle cells ladenwith lipid and surrounded by extra-cellular lipid, collagen, elastin andproteoglycans. The cells plus matrix form a fibrous cap that covers adeeper deposit of cell debris and more extra cellular lipid. The lipidis primarily free and esterified cholesterol. The fibrous plaque formsslowly, and is likely in time to become calcified and necrotic,advancing to the “complicated lesion,” which accounts for the arterialocclusion and tendency toward mural thrombosis and arterial muscle spasmthat characterize advanced atherosclerosis.

Epidemiological evidence has firmly established hyperlipidemia as aprimary risk factor in causing cardiovascular disease (CVD) due toatherosclerosis. In recent years, leaders of the medical profession haveplaced renewed emphasis on lowering plasma cholesterol levels, and lowdensity lipoprotein cholesterol in particular, as an essential step inprevention of CVD. The upper limits of “normal” are now known to besignificantly lower than heretofore appreciated. As a result, largesegments of Western populations are now realized to be at particularlyhigh risk. Such independent risk factors include glucose intolerance,left ventricular hypertrophy, hypertension, and being of the male sex.Cardiovascular disease is especially prevalent among diabetic subjects,at least in part because of the existence of multiple independent riskfactors in this population. Successful treatment of hyperlipidemia inthe general population, and in diabetic subjects in particular, istherefore of exceptional medical importance.

Hypertension (or high blood pressure) is a condition which occurs in thehuman population as a secondary symptom to various other disorders suchas renal artery stenosis, pheochromocytoma or endocrine disorders.However, hypertension is also evidenced in many patients in whom thecausative agent or disorder is unknown. While such “essential”hypertension is often associated with disorders such as obesity,diabetes and hypertriglyceridemia, the relationship between thesedisorders has not been elucidated. Additionally, many patients displaythe symptoms of high blood pressure in the complete absence of any othersigns of disease or disorder.

It is known that hypertension can directly lead to heart failure, renalfailure and stroke (brain hemorrhaging). These conditions are capable ofcausing death in a patient. Hypertension can also contribute to thedevelopment of atherosclerosis and coronary disease. These conditionsgradually weaken a patient and can lead to death.

The exact cause of essential hypertension is unknown, though a number offactors are believed to contribute to the onset of the disease. Amongsuch factors are stress, uncontrolled emotions, unregulated hormonerelease (the renin, angiotensin, aldosterone system), excessive salt andwater due to kidney malfunction, wall thickening and hypertrophy of thevasculature resulting in constricted blood vessels and genetic factors.

The treatment of essential hypertension has been undertaken bearing theforegoing factors in mind. Thus, a broad range of beta-blockers,vasoconstrictors, angiotensin converting enzyme inhibitors and the likehave been developed and marketed as antihypertensives. The treatment ofhypertension utilizing these compounds has proven beneficial in theprevention of short-interval deaths such as heart failure, renal failureand brain hemorrhaging. However, the development of atherosclerosis orheart disease due to hypertension over a long period of time remains aproblem. This implies that although high blood pressure is beingreduced, the underlying cause of essential hypertension is notresponding to this treatment.

Hypertension has been associated with elevated blood insulin levels, acondition known as hyperinsulinemia. Insulin, a peptide hormone whoseprimary actions are to promote glucose utilization, protein synthesisand the formation and storage of neutral lipids, also acts to promotevascular cell growth and increase renal sodium retention, among otherthings. These latter functions can be accomplished without affectingglucose levels and are known causes of hypertension. Peripheralvasculature growth, for example, can cause constriction of peripheralcapillaries while sodium retention increases blood volume. Thus, thelowering of insulin levels in hyperinsulinemics can prevent abnormalvascular growth and renal sodium retention caused by high insulin levelsand thereby alleviate hypertension.

Cardiac hypertrophy is a significant risk factor in the development ofsudden death, myocardial infarction, and congestive heart failure. Thesecardiac events are due, at least in part, to increased susceptibility tomyocardial injury after ischemia and reperfusion that can occur inout-patient as well as perioperative settings. There is an unmet medicalneed to prevent or minimize adverse myocardial perioperative outcomes,particularly perioperative myocardial infarction. Both non-cardiac andcardiac surgery are associated with substantial risks for myocardialinfarction or death. Some 7 million patients undergoing non-cardiacsurgery are considered to be at risk, with incidences of perioperativedeath and serious cardiac complications as high as 20-25% in someseries. In addition, of the 400,000 patients undergoing coronary by-passsurgery annually, perioperative myocardial infarction is estimated tooccur in 5% and death in 1-2%. There is currently no marketed drugtherapy in this area which reduces damage to cardiac tissue fromperioperative myocardial ischemia or enhances cardiac resistance toischemic episodes. Such a therapy is anticipated to be life-saving andreduce hospitalizations, enhance quality of life and reduce overallhealth care costs of high risk patients. The mechanism(s) responsiblefor the myocardial injury observed after ischemia and reperfusion is notfully understood. It has been reported (M. F. Allard, et a., Am. J.Physiol., 267: H66-H74 (1994)) that “pre ischemic glycogen reduction . .. is associated with improved post ischemic left ventricular functionalrecovery in hypertrophied rat hearts”.

In addition to myocardial ischemia, other tissues can undergo ischemiaand be damaged resulting in serious problems for the paitent. Examplesof such tissues include cardiac, brain, liver, kidney, lung, gut,skeletal muscle, spleen, pancreas, nerve, spinal cord, retina tissue,the vasculature, or intestinal tissue.

Hepatic glucose production is an important target for NIDDM therapy. Theliver is the major regulator of plasma glucose levels in the postabsorptive (fasted) state, and the rate of hepatic glucose production inNIDDM patients is significantly elevated relative to normal individuals.Likewise, in the postprandial (fed) state, where the liver has aproportionately smaller role in the total plasma glucose supply, hepaticglucose production is abnormally high in NIDDM patients.

Glycogenolysis is an important target for interruption of hepaticglucose production. The liver produces glucose by glycogenolysis(breakdown of the glucose polymer glycogen) and gluconeogenesis(synthesis of glucose from 2- and 3-carbon precursors). Several lines ofevidence indicate that glycogenolysis may make an important contributionto hepatic glucose output in NIDDM. First, in normal post absorptiveman, up to 75% of hepatic glucose production is estimated to result fromglycogenolysis. Second, patients having liver glycogen storage diseases,including Hers' disease (glycogen phosphorylase deficiency), displayepisodic hypoglycemia. These observations suggest that glycogenolysismay be a significant process for hepatic glucose production.

Glycogenolysis is catalyzed in liver, muscle, and brain bytissue-specific isoforms of the enzyme glycogen phosphorylase. Thisenzyme cleaves the glycogen macromolecule to release glucose-1-phosphateand a new shortened glycogen macromolecule. Several types of glycogenphosphorylase inhibitors have been reported to date: glucose and glucoseanalogs [Martin, J. L. et al., Biochemistry, 30:10101 (1991)]; caffeineand other purine analogs [Kasvinsky, P. J. et al., J. Biol. Chem., 253:3343-3351 and 9102-9106 (1978)]; substitutedN-(indole-2-carbonyl)-amides [PCT Publication Number WO 96/39385]; andsubstituted N-(indole-2-carbonyl)-glycinamides [PCT Publication NumberWO 96/39384]. These compounds and glycogen phosphorylase inhibitors ingeneral, have been postulated to be of use for the treatment of NIDDM bydecreasing hepatic glucose production and lowering glycemia. [Blundell,T. B. et al., Diabetologia, 35: Suppl. 2, 569-576 (1992) and Martin etal., Biochemistry, 30: 10101 (1991)].

Myocardial ischemic injury can occur in outpatient as well as inperioperative settings and can lead to the development of sudden death,myocardial infarction or congestive heart failure. There is an unmetmedical need to prevent or minimize myocardial ischemic injury,particularly perioperative myocardial infarction. Such a therapy isanticipated to be life-saving and reduce hospitalizations, enhancequality of life and reduce overall health care costs of high riskpatients.

Although there are a variety of hyperglycemia, hypercholesterolemia,hypertension, hyperlipidemia, atherosclerosis and tissue ischemiatherapies, there is a continuing need and a continuing search in thisfield of art for alternative therapies.

SUMMARY OF THE INVENTION

The present invention provides compounds of Formula I:

stereoisomers, pharmaceutically acceptable salts and prodrugs thereof,and pharmaceutically acceptable salts of the prodrugs,

wherein

Q is aryl, substitued aryl, heteroaryl, or substitued heteroaryl;

each Z and X are independently (C, CH or CH₂), N, O or S;

X¹ is NR^(a), —CH₂—, O or S;

each - - - - is independently a bond or is absent, provided thatboth - - - - are not simlutaneously bonds;

R¹ is hydrogen, halogen, —OC₁-C₈alkyl, —SC¹⁻-C₈alkyl, —C₁-C₈alkyl, —CF₃,—NH₂, —NHC₁-C₈alkyl, —N(C₁-C₈alkyl)₂, —NO₂, —CN, —CO₂H, —CO₂C₁-C₈alkyl,—C₂-C₈alkenyl, or —C₂-C₈alkynyl;

each R^(a) and R^(b) is independently hydrogen or —C₁-C₈alkyl;

Y is

 or absent;

R² and R³ are independently hydrogen, halogen, —C₁-C₈alkyl, —CN,—C≡C—Si(CH₃)₃, —OC₁-C₈alkyl, —SC₁-C₈alkyl, —CF₃, —NH₂, —NHC₁-C₈alkyl,—N(C₁-C₈alkyl)₂, —NO₂, —CO₂H, —CO₂C₁-C₈alkyl, —C₂-C₈alkenyl, or—C₂-C₈alkynyl, or R² and R³ together with the atoms on the ring to whichthey are attached form a five or six membered ring containing from 0 to3 heteroatoms and from 0 to 2 double bonds;

R⁴ is —C(═O)-A;

A is —NR^(d)R^(d), —NR^(a)CH₂CH₂OR^(a),

 each R^(d) is independently hydrogen, C₁-C₈alkyl, C₁-C₈alkoxy, aryl,substituted aryl, heteroaryl, or substituted heteroaryl;

each R^(c) is independently hydrogen, —C(═O)OR^(a), —OR^(a), —SR^(a), or—NR^(a)R^(a); and each n is independently 1-3.

In a preferred embodiment of the compounds of Formula I, R^(b) and R¹are hydrogen.

In another preferred embodiment of the compounds of Formula I,

R^(b) is hydrogen;

R¹ is hydrogen;

Y is

and A is

In another preferred embodiment of the compounds of Formula I,

R^(b) is hydrogen;

R¹ is hydrogen;

Y is absent; and

A is

In another preferred embodiment of the compounds of Formula I,

R^(b) is hydrogen;

R¹ is hydrogen;

Z is C;

X is O or S;

Y is absent;

A is

R² is hydrogen; and

R³ is hydrogen, halogen or methyl.

In another preferred embodiment of the compounds of Formula I,

Q is phenyl and A is

In another preferred embodiment, the invention provides compounds ofFormula I

stereoisomers, pharmaceutically acceptable salts and prodrugs thereof,and pharmaceutically acceptable salts of the prodrugs,

wherein

Q is phenyl;

(Z is Sand X is C), (Z is C and X is S), or (Z is C and X is O);

each - - - - is independently a bond or is absent, provided thatboth - - - - are not simultaneously bonds;

R¹ is hydrogen or halogen,

each R^(a) and R^(b) is independently hydrogen or C₁-C₈alkyl;

Y is

 or absent;

R² and R³ are independently hydrogen, halogen, C₁-C₈alkyl, —CN,—C≡CSi(CH₃)₃, or C₂-C₈alkynyl, or R² and R³ together with the atoms onthe ring to which they are attached form a five or six membered ringcontaining from 0 to 3 heteroatoms and from 0 to 2 double bonds;

R⁴ is —C(═O)-A;

A is —NR^(d)R^(d),

each R^(d) is independently C₀-C₈alkyl;

each R^(c) is independently hydrogen, —OH, or —C(═O))C₁-C₈alkyl;

each n is independently 1-3.

In another preferred embodiment of the compounds of Formula I,

Also provided are pharmaceutical compositions comprising a compound ofFormula I, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating or preventing atherosclerosis, themethods comprising the step of administering to a patient havingatherosclerosis or at risk of having atherosclerosis a therapeuticallyeffective amount of a compound of Formula I, stereoisomers,pharmaceutically acceptable salts and prodrugs thereof, andpharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating diabetes, the methods comprisingthe step of administering to a patient having diabetes a therapeuticallyeffective amount of a compound of Formula I, stereoisomers,pharmaceutically acceptable salts and prodrugs thereof, andpharmaceutically acceptable salts of the prodrug.

In a preferred embodiment of the methods of treating diabetes, thediabetes is non-insulin dependent diabetes mellitus (Type II).

In another preferred embodiment of the methods of treating diabetes, thediabetes is insulin dependent diabetes mellitus (Type I).

Also provided are methods of treating insulin resistance, the methodscomprising the step of administering to a patient having insulinresistance a therapeutically effective amount of a compound of FormulaI, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating diabetic neuropathy, the methodscomprising the step of administering to a patient having diabeticneuropathy a therapeutically effective amount of a compound of FormulaI, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating diabetic nephropathy, the methodscomprising the step of administering to a patient having diabeticnephropathy a therapeutically effective amount of a compound of FormulaI, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating diabetic retinopathy, the methodscomprising the step of administering to a patient having diabeticretinopathy a therapeutically effective amount of a compound of FormulaI, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating cataracts, the methods comprisingthe step of administering to a patient having cataracts atherapeutically effective amount of a compound of Formula I,stereoisomers, pharmaceutically acceptable salts and prodrugs thereof,and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating or preventinghypercholesterolemia, the methods comprising the step of administeringto a patient having hypercholesterolemia or at risk of havinghypercholesterolemia a therapeutically effective amount of a compound ofFormula I, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating or preventinghypertriglyceridemia, the methods comprising the step of administeringto a patient having hypertriglyceridemia or at risk of havinghypertriglyceridemia a therapeutically effective amount of a compound ofFormula I, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating or preventing hyperlipidemia, themethods comprising the step of administering to a patient havinghyperlipidemia or at risk of having hyperlipidemia a therapeuticallyeffective amount of a compound of Formula I, stereoisomers,pharmaceutically acceptable salts and prodrugs thereof, andpharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating hyperglycemia, the methodscomprising the step of administering to a patient having hyperglycemiaor at risk of having hyperglycemia therapeutically effective amount of acompound of Formula I, stereoisomers, pharmaceutically acceptable saltsand prodrugs thereof, and pharmaceutically acceptable salts of theprodrugs.

Also provided are methods of treating hypertension, the methodscomprising the step of administering to a patient having hypertension orat risk of having hypertension a therapeutically effective amount of acompound of Formula I, stereoisomers, pharmaceutically acceptable saltsand prodrugs thereof, and pharmaceutically acceptable salts of theprodrugs.

Also provided are methods of treating or preventing tissue ischemia, themethods comprising the step of administering to a patient having tissueischemia or at risk of having tissue ischemia a therapeuticallyeffective amount of a compound of Formula I, stereoisomers,pharmaceutically acceptable salts and prodrugs thereof, andpharmaceutically acceptable salts of the prodrugs.

Also provided are methods of treating or preventing myocardial ischemia,the methods comprising the step of administering to a patient havingmyocardial ischemia or at risk of having myocardial ischemia atherapeutically effective amount of a compound of Formula I,stereoisomers, pharmaceutically acceptable salts and prodrugs thereof,and pharmaceutically acceptable salts of the prodrugs.

Also provided are methods of inhibiting glycogen phosphorylase, themethods comprising the step of administering to a patient in need ofglycogen phosphorylase inhibition, a glycogen phosphorylase inhibitingamount of a compound of Formula I, stereoisomers, pharmaceuticallyacceptable salts and prodrugs thereof, and pharmaceutically acceptablesalts of the prodrugs.

The present invention provides the compounds:

6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

2-bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

2-methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

(±)-2-methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[1-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2,4-dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

(±)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[1-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

(±)-2-bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[1-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2,4-dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-Cyano-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide;

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-morpholin-4-yl-2-oxo-ethyl]-amide;

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-dimethylcarbamoyl-2-phenyl-ethyl]-amide;

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(1,1-dioxo-1-thiazolidin-3-yl)-2-oxo-ethyl]-amide;

1-{(2S)-[(2-chloro-6H-thieno[2,3-b]pyrrole-5-carbonyl)-amino-3-phenyl-propionyl}-piperidine-4-carboxylicacid ethyl ester;

2-bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide;

2-methyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-trimethylsilanylethynyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide;

2-ethynyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide;

2-fluoro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-Cyano-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide;

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

1-{(2S)-[(2-chloro-6H-thieno[2,3-b]pyrrole-5-carbonyl)-amino]-3-phenyl-propionyl}-piperidine-4-carboxylicacid;

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

3-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

3-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

2-Cyano-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-Cyano-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

3-bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

3-bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

4H-1,7-dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

4H-1,7-dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;

2-methylsulfanyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide;

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(1,1-dioxo-1-thiazolidin-3-yl)-2-oxo-ethyl]-amide;

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-morpholin-4-yl-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3S,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4R)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;and

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(4-hydroxy-piperidin-1-yl)-2-oxo-ethyl]-amide, andstereoisomers, pharmaceutically acceptable salts and prodrugs of thecompounds, and pharmaceutically acceptable salts of the prodrugs.

Also provided are kits for the treatment of diabetes, insulinresistance, diabetic neuropathy, diabetic nephropathy, diabeticretinopathy, or cataracts in a patient having diabetes, insulinresistance, diabetic neuropathy, diabetic nephropathy, diabeticretinopathy, or cataracts, the kits comprising:

a) a first pharmaceutical composition comprising a compound of FormulaI, stereoisomers, pharmaceutically acceptable salts and prodrugs of thecompounds of Formula I, and pharmaceutically acceptable salts of theprodrugs;

b) a second pharmaceutical composition comprising a second compounduseful for the treatment of diabetes, insulin resistance, diabeticneuropathy, diabetic nephropathy, diabetic retinopathy, or cataracts;and

c) a container for containing the first and second compositions.

In a preferred embodiment of the kits, the second compound is selectedfrom: insulin and insulin analogs;

GLP-1 (7-37) (insulinotropin) and GLP-1 (7-36)—NH₂;

sulfonylureas and analogs;

biguanides;

α2-antagonists;

imidazolines;

glitazones (thiazolidenediones);

PPAR-gamma agonists;

fatty acid oxidation inhibitors;

α-glucosidase inhibitors;

β-agonists;

phosphodiesterase Inhibitors;

lipid-lowering agents:

antiobesity agents

vanadate, vanadium complexes and peroxovanadium complexes;

amylin antagonists;

glucagon antagonists;

gluconeogenesis inhibitors;

somatostatin analogs and antagonists; and

antilipolytic agents.

In another preferred embodiment of the kits, the second compound isselected from LysPro insulin, GLP-1 (7-37) (insulinotropin), GLP-1(7-36)—NH₂, chlorpropamide, glibenclamide, tolbutamide, tolazamide,acetohexamide, glypizide, glimepiride, repaglinide, meglitinide;mefformin, phenformin, buformin, midaglizole, isaglidole, deriglidole,idazoxan, efaroxan, fluparoxan, linogliride, ciglitazone, pioglitazone,englitazone, troglitazone, darglitazone, rosiglitazone, clomoxir,etomoxir, acarbose, miglitol, emiglitate, voglibose, MDL-25,637,camiglibose, MDL-73,945, BRL 35135, BRL 37344, Ro 16-8714, ICI D7114, CL316,243, L-386,398; benfluorex, fenfluramine, Naglivan®, acipimox, WAG994, Symlin™, AC2993 and nateglinide.

In still another preferred embodiment of the kits, the second compoundis selected from insulin, sulfonylureas, biguanides, andthiazolidinediones.

Also provided are kits for the treatment of diabetes, insulinresistance, diabetic neuropathy, diabetic nephropathy, diabeticretinopathy, cataracts, hyperglycemia, hypercholesterolemia,hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, ortissue ischemia in a patient having diabetes, insulin resistance,diabetic neuropathy, diabetic nephropathy, diabetic retinopathy,cataracts, hyperglycemia, hypercholesterolemia, hypertension,hyperinsulinemia, hyperlipidemia, atherosclerosis, or tissue ischemia,the kits comprising:

a) a first pharmaceutical composition comprising a compound of FormulaI, stereoisomers, pharmaceutically acceptable salts and prodrugs of thecompounds of Formula I, and pharmaceutically acceptable salts of theprodrugs;

b) a second pharmaceutical composition comprising a second compounduseful for the treatment of diabetes, insulin resistance, diabeticneuropathy, diabetic nephropathy, diabetic retinopathy, cataracts,hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia,hyperlipidemia, atherosclerosis, or tissue ischemia; and

c) a container for containing the first and second compositions.

Also provided are methods of treating diabetes, insulin resistance,diabetic neuropathy, diabetic nephropathy, diabetic retinopathy,cataracts, hyperglycemia, hypercholesterolemia, hypertension,hyperinsulinemia, hyperlipidemia, atherosclerosis, or tissue ischemia,the method comprising the step of administering to a patient havingdiabetes, insulin resistance, diabetic neuropathy, diabetic nephropathy,diabetic retinopathy, cataracts, hyperglycemia, hypercholesterolemia,hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, ortissue ischemia, a therapeutically effective amount of a compound ofFormula I, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs incombination with at least one additional compound useful for thetreatment of diabetes, insulin resistance, diabetic neuropathy, diabeticnephropathy, diabetic retinopathy, cataracts, hyperglycemia,hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia,atherosclerosis, or tissue ischemia.

Also provided are pharmaceutical compositions comprising a compound ofFormula I, stereoisomers, pharmaceutically acceptable salts and prodrugsthereof, and pharmaceutically acceptable salts of the prodrugs and atleast one additional compound useful to treat diabetes, insulinresistance, diabetic neuropathy, diabetic nephropathy, diabeticretinopathy, cataracts, hyperglycemia, hypercholesterolemia,hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, ortissue ischemia.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds of Formula I, stereoisomersof compounds of Formula I, pharmaceutically acceptable salts ofcompounds of Formula I, prodrugs of compounds of Formula I, andpharmaceutically acceptable salts of the prodrugs of compounds ofFormula I. The invention also relates to methods of treatment ofdiabetes, insulin resistance, diabetic neuropathy, diabetic nephropathy,diabetic retinopathy, cataracts, hyperglycemia, hypercholesterolemia,hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, andtissue ischemia, particularly myocardial ischemia, and topharmaceutically acceptable compositions comprising a compound ofFormula I, stereoisomers of compounds of Formula I, pharmaceuticallyacceptable salts of compounds of Formula I, prodrugs of compounds ofFormula I, and pharmaceutically acceptable salts of the prodrugs ofcompounds of Formula I.

Certain terms that are used in this application are defined below.

The term “alkyl” means a straight or branched chain hydrocarbon.Representative examples of alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, and hexyl.Preferred alkyl groups are C₁-C₈alkyl.

The term “alkoxy” means an alkyl group bonded to an oxygen atom.Representative examples of alkoxy groups include methoxy, ethoxy,tert-butoxy, propoxy, and isobutoxy. Preferred alkoxy groups areC₁-C₈alkoxy.

The term “halogen” means chlorine, fluorine, bromine or iodine.

The term “alkenyl” means a branched or straight chain hydrocarbon havingone or more carbon-carbon double bonds.

The term “alkynyl” means a branched or straight chain hydrocarbon havingone or more carbon-carbon triple bonds.

The term “cycloalkyl” means a cyclic, hydrocarbon. Examples ofcycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl. Preferred cycloalkyl groups areC₃-C₈Cyloalkyl. It is also possible for the cycloalkyl group to have oneor more double bonds, but is not aromatic. Examples of cycloalkyl groupshaving double bonds include cyclopentenyl, cyclohexenyl,cyclohexadienyl, cyclobutadienyl, and the like.

The term “perfluoroalkyl” means an alkyl group in which all of thehydrogen atoms have been replaced with fluorine atoms.

The term “acyl” means a group derived from an organic acid (—COOH) byremoval of the hydroxy group (—OH).

The term “aryl” means a cyclic, aromatic hydrocarbon. Examples of arylgroups include phenyl and naphthyl.

The term “heteroatom” includes oxygen, nitrogen, sulfur, andphosphorous.

The term “heteroaryl” means a cyclic, aromatic hydrocarbon in which oneor more carbon atoms have been replaced with a heteroatom. If theheteroaryl group contains more than one heteroatom, the heteroatoms maybe the same or different. Examples of heteroaryl groups include pyridyl,pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl, indolizinyl,triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, isothiazolyl, andbenzo[b]thienyl. Preferred heteroaryl groups are five or six memberedrings and contain from one to three heteroatoms.

The term “heterocycloalkyl” means a cycloalkyl group in which one ormore of the carbon atoms has been replaced with a heteroatom. If theheterocycloalkyl group contains more than one heteroatom, theheteroatoms may be the same or different. Examples of heterocycloalkylgroups include tetrahydrofuryl, morpholinyl, piperazinyl, piperadyl, andpyrrolidinyl. Preferred heterocycloalkyl groups are five or six memberedrings and contain from one to three heteroatoms. It is also possible forthe heterocycloalkyl group to have one or more double bonds, but is notaromatic. Examples of heterocycloalkyl groups containing double bondsinclude dihydrofuran, and the like.

It is also noted that the cyclic ring groups, i.e., aryl, heteroaryl,cycloalkyl, heterocycloalkyl, can comprise more than one ring. Forexample, the naphthyl group is a fused bicyclic ring system. It is alsointended that the present invention include ring groups that havebridging atoms, or ring groups that have a spiro orientation.

Representative examples of five to six membered aromatic rings,optionally having one or two heteroatoms, are phenyl, furyl, thienyl,pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,isothiazolyl, pyridinyl, pyridiazinyl, pyrimidinyl, and pyrazinyl.

Representative examples of partially saturated, fully saturated or fullyunsaturated five to eight membered rings, optionally having one to threeheteroatoms, are cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl andphenyl. Further exemplary five membered rings are furyl, thienyl,pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl,oxazolyl, thiazolyl, imidazolyl, 2H-imidazolyl, 2-imidazolinyl,imidazolidinyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl,isothiazolyl, 1,2-dithiolyl, 1,3-dithiolyl, 3H-1,2-oxathiolyl,1,2,3-oxadizaolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,4-trizaolyl, 1,3,4-thiadiazolyl,3H-1,2,3-dioxazolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl,1,3,4-dioxazolyl, 5H-1,2,5-oxathiazolyl, and 1,3-oxathiolyl.

Further exemplary six membered rings are 2H-pyranyl, 4H-pyranyl,pyridinyl, piperidinyl, 1,2-dioxinyl, 1,3-dioxinyl, 1,4-dioxanyl,morpholinyl, 1,4-dithianyl, thiomorpholinyl, pyridazinyl, pyrimidinyl,pyrazinyl, piperazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl,1,2,3-triazinyl, 1,3,5-trithianyl, 4H-1,2-oxazinyl, 2H-1,3-oxazinyl,6H-1,3-oxazinyl, 6H-1,2-oxazinyl, 1,4-oxazinyl, 2H-1,2-oxazinyl,4H-1,4-oxazinyl, 1,2,5-oxathiazinyl, 1,4-oxazinyl, o-isoxazinyl,p-isoxazinyl, 1,2,5-oxathiazinyl, 1,2,6-oxathiazinyl, and1,4,2-oxadiazinyl.

Further exemplary seven membered rings are azepinyl, oxepinyl, thiepinyland 1,2,4-triazepinyl.

Further exemplary eight membered rings are cyclooctyl, cyclooctenyl andcyclooctadienyl.

Exemplary bicyclic rings consisting of two fused partially saturated,fully saturated or fully unsaturated five and/or six membered rings,taken independently, optionally having one to four heteroatoms areindolizinyl, indolyl, isoindolyl, indolinyl, cyclopenta(b)pyridinyl,pyrano(3,4-b)pyrrolyl, benzofuryl, isobenzofuryl, benzo(b)thienyl,benzo(c)thienyl, 1H-indazolyl, indoxazinyl, benzoxazolyl, anthranilyl,benzimidazolyl, benzthiazolyl, purinyl, quinolinyl, isoquinolinyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, indenyl, isoindenyl, naphthyl,tetralinyl, decalinyl, 2H-1-benzopyranyl, pyrido(3,4-b)-pyridinyl,pyrido(3,2-b)-pyridinyl, pyrido(4,3-b)-pyridinyl, 2H-1,3-benzoxazinyl,2H-1,4-benzoxazinyl, 1H-2,3-benzoxazinyl, 4H-3,1-benzoxazinyl,2H-1,2-benzoxazinyl and 4H-1,4-benzoxazinyl.

A cyclic ring group may be bonded to another group in more than one way.If no particular bonding arrangement is specified, then all possiblearrangements are intended. For example, the term “pyridyl” includes 2-,3-, or 4-pyridyl, and the term “thienyl” includes 2-, or 3-thienyl.

The term “substituted” means that a hydrogen atom on an organic moleculehas been replaced with a different atom or with a molecule. The atom ormolecule replacing the hydrogen atom is called a substituent. Examplesof suitable substituents include, halogens, —OC₁-C₈alkyl, —C₁-C₈alkyl,—CF₃, —NH₂, —NHC₁-C₈alkyl, —N(C₁-C₈alkyl)₂, —NO₂, —CN, —CO₂H,—CO₂C₁-C₈alkyl, and the like.

The symbol “-” represents a covalent bond.

The term “therapeutically effective amount” means an amount of acompound that ameliorates, attenuates, or eliminates one or more symptomof a particular disease or condition or prevents or delays the onset ofone of more symptom of a particular disease or condition.

The term “patient” means animals, such as dogs, cats, cows, horses,sheep, and humans. Particularly preferred patients are mammals. The termpatient includes males and females.

The term “pharmaceutically acceptable” means that the carrier, diluent,excipients, and/or salt must be compatible with the other ingredients ofthe formulation, and not deleterious to the patient.

The phrases “a compound of the present invention, compounds of thepresent invention, a compound of Formula I, or a compound in accordancewith Formula I” and the like, includes the stereoisomers of thecompound(s), pharmaceutically acceptable salts of the compound(s),prodrugs of the compound(s), and pharmaceutically acceptable salts ofthe prodrugs.

The terms “reaction-inert solvent” or “inert solvent” refer to a solventor mixture of solvents that does not interact with starting materials,reagents, intermediates or products in a manner that adversely affectsthe desired product.

The terms “treating”, “treat” or “treatment” include preventative (e.g.,prophylactic) and palliative treatment.

The term “glycogen phosphorylase inhibitor” refers to any substance oragent or any combination of substances and/or agents that reduces,retards, or eliminates the enzymatic action of glycogen phosphorylase.The currently known enzymatic action of glycogen phosphorylase is thedegradation of glycogen by catalysis of the reversible reaction of aglycogen macromolecule and inorganic phosphate to glucose-1-phosphateand a glycogen macromolecule which is one glucosyl residue shorter thanthe original glycogen macromolecule (forward direction ofglycogenolysis).

A patient in need of glycogen phosphorylase inhibition is a patienthaving a disease or condition in which glycogen phosphorylase plays arole in the disease of condition. Examples of patients in need ofglycogen phoshphorylase inhibition include patients having diabetes(including Type I and Type II, impaired glucose tolerance, insulinresistance, and the diabetic complications, such a nephropathy,retinopathy, neuropathy and cataracts), hyperglycemia,hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia,atherosclerosis and tissue ischemia.

The characteristics of patients at risk of having atherosclerosis arewell known to those skilled in the art and include, patients who have afamily history of cardiovascular disease, including hypertension andatherosclerosis, obese patients, patient who exercise infrequently,patients with hypercholesterolemia, patients having high levels of lowdensity lipoprotein (LDL), patients having low levels of high densitylipoprotein (HDL), and the like.

Patients at risk of having myocardial ischemia and other tissueischemias are also well known to those skilled in the art and includepatients undergoing or having undergone surgery, trauma or great stress.

The compounds of the present invention are administered to a patient ina therapeutically effective amount. The compounds can be administeredalone or as part of a pharmaceutically acceptable composition orformulation. In addition, the compounds or compositions can beadministered all at once, as for example, by a bolus injection, multipletimes, such as by a series of tablets, or delivered substantiallyuniformly over a period of time, as for example, using transdermaldelivery. It is also noted that the dose of the compound can be variedover time.

In addition, the compounds of the present invention can be administeredalone, in combination with other compounds of the present invention, orwith other pharmaceutically active compounds. The other pharmaceuticallyactive compounds can be intended to treat the same disease or conditionas the compounds of the present invention or a different disease orcondition. If the patient is to receive or is receiving multiplepharmaceutically active compounds, the compounds can be administeredsimultaneously, or sequentially. For example, in the case of tablets,the active compounds may be found in one tablet or in separate tablets,which can be administered at once or sequentially in any order. Inaddition, it should be recognized that the compositions may be differentforms. For example, one or more compound may be delivered via a tablet,while another is administered via injection or orally as a syrup. Allcombinations, delivery methods and administration sequences arecontemplated.

Since one aspect of the present invention contemplates the treatment ofthe disease/conditions with a combination of pharmaceutically activeagents that may be administered separately, the invention furtherrelates to combining separate pharmaceutical compositions in kit form.The kit comprises two separate pharmaceutical compositions: a compoundof the present invention, and a second pharmaceutical compound. The kitcomprises a container for containing the separate compositions such as adivided bottle or a divided foil packet. Additional examples ofcontainers include syringes, boxes, bags, and the like. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

An example of such a kit is a so-called blister pack. Blister packs arewell known in the packaging industry and are being widely used for thepackaging of pharmaceutical unit dosage forms (tablets, capsules, andthe like). Blister packs generally consist of a sheet of relativelystiff material covered with a foil of a preferably transparent plasticmaterial. During the packaging process recesses are formed in theplastic foil. The recesses have the size and shape of the tablets orcapsules to be packed. Next, the tablets or capsules are placed in therecesses and the sheet of relatively stiff material is sealed againstthe plastic foil at the face of the foil which is opposite from thedirection in which the recesses were formed. As a result, the tablets orcapsules are sealed in the recesses between the plastic foil and thesheet. Preferably the strength of the sheet is such that the tablets orcapsules can be removed from the blister pack by manually applyingpressure on the recesses whereby an opening is formed in the sheet atthe place of the recess. The tablet or capsule can then be removed viasaid opening.

It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, . . . etc. . . . . Second Week, Monday, Tuesday, . . . ” etc.Other variations of memory aids will be readily apparent. A “daily dose”can be a single tablet or capsule or several pills or capsules to betaken on a given day. Also, a daily dose of a compound of the presentinvention can consist of one tablet or capsule, while a daily dose ofthe second compound can consist of several tablets or capsules and viceversa. The memory aid should reflect this and aid in correctadministration of the active agents.

In another specific embodiment of the invention, a dispenser designed todispense the daily doses one at a time in the order of their intendeduse is provided. Preferably, the dispenser is equipped with amemory-aid, so as to further facilitate compliance with the regimen. Anexample of such a memory-aid is a mechanical counter which indicates thenumber of daily doses that has been dispensed. Another example of such amemory-aid is a battery-powered micro-chip memory coupled with a liquidcrystal readout, or audible reminder signal which, for example, readsout the date that the last daily dose has been taken and/or reminds onewhen the next dose is to be taken.

The compounds of the present invention and other pharmaceutically activeagents, if desired, can be administered to a patient either orally,rectally, parenterally, (for example, intravenously, intramuscularly, orsubcutaneously) intracisternally, intravaginally, intraperitoneally,intravesically, locally (for example, powders, ointments or drops), oras a buccal or nasal spray.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. Microorganism contaminationcan be prevented by adding various antibacterial and antifungal agents,for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.It may also be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption ofinjectable pharmaceutical compositions can be brought about by the useof agents delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage forms, the active compoundis admixed with at least one inert customary excipient (or carrier) suchas sodium citrate or dicalcium phosphate or (a) fillers or extenders, asfor example, starches, lactose, sucrose, mannitol, and silicic acid; (b)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, as forexample, glycerol; (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, and sodium carbonate; (e) solution retarders, as forexample, paraffin; (f) absorption accelerators, as for example,quaternary ammonium compounds; (g) wetting agents, as for example, cetylalcohol and glycerol monostearate; (h) adsorbents, as for example,kaolin and bentonite; and (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules, and tablets, thedosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be used as fillers in softand hard filled gelatin capsules using such excipients as lactose ormilk sugar, as well as high molecular weight polyethylene glycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may also containopacifying agents, and can also be of such composition that they releasethe active compound or compounds in a certain part of the intestinaltract in a delayed manner. Examples of embedding compositions that canbe used are polymeric substances and waxes. The active compounds canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage form may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, and sesame seed oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compound, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, and tragacanth, or mixtures ofthese substances, and the like.

Compositions for rectal administration are preferable suppositories,which can be prepared by mixing the compounds of the present inventionwith suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax, which are solid atordinary room temperature, but liquid at body temperature, andtherefore, melt in the rectum or vaginal cavity and release the activecomponent.

Dosage forms for topical administration of a compound of the presentinvention include ointments, powders, sprays and inhalants. The activecompound or compounds are admixed under sterile condition with aphysiologically acceptable carrier, and any preservatives, buffers, orpropellants that may be required. Opthalmic formulations, eye ointments,powders, and solutions are also contemplated as being within the scopeof this invention.

The compounds of the present invention can be administered to a patientat dosage levels in the range of about 0.1 to about 3,000 mg per day.For a normal adult human having a body weight of about 70 kg, a dosagein the range of about 0.01 to about 100 mg per kilogram body weight istypically sufficient. The specific dosage and dosage range that can beused depends on a number of factors, including the requirements of thepatient, the severity of the condition or disease being treated, and thepharmacological activity of the compound being administered. Thedetermination of dosage ranges and optimal dosages for a particularpatient is well within the ordinary skill in the art.

The following paragraphs describe exemplary formulations, dosages etc.useful for non-human animals. The administration of a compound of thepresent invention can be effected orally or non-orally, for example byinjection. An amount of a compound of the present invention isadministered such that an effective dose is received, generally a dailydose which, when administered orally to an animal is usually between0.01 and 100 mg/kg of body weight, preferably between 0.1 and 50 mg/kgof body weight. Conveniently, the medication can be carried in thedrinking water so that a therapeutic dosage of the agent is ingestedwith the daily water supply. The agent can be directly metered intodrinking water, preferably in the form of a liquid, water-solubleconcentrate (such as an aqueous solution of a water soluble salt).Conveniently, the active ingredient can also be added directly to thefeed, as such, or in the form of an animal feed supplement, alsoreferred to as a premix or concentrate. A premix or concentrate oftherapeutic agent in a carrier is more commonly employed for theinclusion of the agent in the feed. Suitable carriers are liquid orsolid, as desired, such as water, various meals such as alfalfa meal,soybean meal, cottonseed oil meal, linseed oil meal, corncob meal andcorn meal, molasses, urea, bone meal, and mineral mixes such as arecommonly employed in poultry feeds. A particularly effective carrier isthe respective animal feed itself; that is, a small portion of suchfeed. The carrier facilitates uniform distribution of the activematerials in the finished feed with which the premix is blended. It isimportant that the compound be thoroughly blended into the premix and,subsequently, the feed. In this respect, the agent may be dispersed ordissolved in a suitable oily vehicle such as soybean oil, corn oil,cottonseed oil, and the like, or in a volatile organic solvent and thenblended with the carrier. It will be appreciated that the proportions ofactive material in the concentrate are capable of wide variation sincethe amount of agent in the finished feed may be adjusted by blending theappropriate proportion of premix with the feed to obtain a desired levelof therapeutic agent.

High potency concentrates may be blended by the feed manufacturer withproteinaceous carrier such as soybean oil meal and other meals, asdescribed above, to produce concentrated supplements which are suitablefor direct feeding to animals. In such instances, the animals arepermitted to consume the usual diet. Alternatively, such concentratedsupplements may be added directly to the feed to produce a nutritionallybalanced, finished feed containing a therapeutically effective level ofa compound according to the invention. The mixtures are thoroughlyblended by standard procedures, such as in a twin shell blender, toensure homogeneity.

If the supplement is used as a top dressing for the feed, it likewisehelps to ensure uniformity of distribution of the active material acrossthe top of the dressed feed.

Drinking water and feed effective for increasing lean meat depositionand for improving lean meat to fat ratio are generally prepared bymixing a compound of the invention with a sufficient amount of animalfeed to provide from about 10⁻³ to about 500 ppm of the compound in thefeed or water.

The preferred medicated swine, cattle, sheep and goat feed generallycontain from about 1 to about 400 grams of active ingredient per ton offeed, the optimum amount for these animals usually being about 50 toabout 300 grams per ton of feed.

The preferred poultry and domestic pet feeds usually contain about 1 toabout 400 grams and preferably about 10 to about 400 grams of activeingredient per ton of feed.

For parenteral administration in animals, the compounds of the presentinvention may be prepared in the form of a paste or a pellet andadministered as an implant, usually under the skin of the head or ear ofthe animal.

In general, parenteral administration involves injection of a sufficientamount of a compound of the present invention to provide the animal withabout 0.01 to about 100 mg/kg/day of body weight of the activeingredient. The preferred dosage for poultry, swine, cattle, sheep,goats and domestic pets is in the range of from about 0.1 to about 50mg/kg/day.

Paste formulations can be prepared by dispersing the active compound ina pharmaceutically acceptable oil such as peanut oil, sesame oil, cornoil or the like.

Pellets containing an effective amount of a compound of the presentinvention can be prepared by admixing a compound of the presentinvention with a diluent such as carbowax, carnuba wax, and the like,and a lubricant, such as magnesium or calcium stearate, can be added toimprove the pelleting process.

It is, of course, recognized that more than one pellet may beadministered to an animal to achieve the desired dose level. Moreover,it has been found that implants may also be made periodically during theanimal treatment period in order to maintain the proper active agent inthe level animal's body.

The term pharmaceutically acceptable salts, esters, amides, or prodrugsmeans the carboxylate salts, amino acid addition salts, esters, amides,and prodrugs of the compounds of the present invention that are, withinthe scope of sound medical judgment, suitable for use with patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the present invention.

The term “salts” refers to inorganic and organic salts of compounds ofthe present invention. The salts can be prepared in situ during thefinal isolation and purification of a compound, or by separatelyreacting a purified compound in its free base form with a suitableorganic or inorganic acid and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, palmitiate, stearate, laurate,borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,lactobionate, and laurylsulphonate salts, and the like. The salts mayinclude cations based on the alkali and alkaline earth metals, such assodium, lithium, potassium, calcium, magnesium, and the like, as well asnon-toxic ammonium, quaternary ammonium, and amine cations including,but not limited to, ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. See, for example, S. M. Berge, et al., “PharmaceuticalSalts,” J Pharm Sci, 66: 1-19 (1977).

Examples of pharmaceutically acceptable, non-toxic esters of thecompounds of the present invention, if applicable, include C₁-C₈ alkylesters. Acceptable esters also include C₅-C₇Cycloalkyl esters, as wellas arylalkyl esters such as benzyl. C₁-C₄ alkyl esters are preferred.Esters of compounds of the present invention may be prepared accordingto methods that are well known in the art.

Examples of pharmaceutically acceptable non-toxic amides of thecompounds of the present invention include amides derived from ammonia,primary C₁-C₈alkyl amines, and secondary C₁-C₈ dialkyl amines. In thecase of secondary amines, the amine may also be in the form of a 5 or 6membered heterocycloalkyl group containing at least one nitrogen atom.Amides derived from ammonia, C₁-C₃ primary alkyl amines, and C₁-C₂dialkyl secondary amines are preferred. Amides of the compounds of thepresent invention may be prepared according to methods well known tothose skilled in the art.

The term “prodrug” means compounds that are transformed in vivo to yielda compound of Formula I. The transformation may occur by variousmechanisms, such as through hydrolysis in blood. A discussion of the useof prodrugs is provided by T. Higuchi and W. Stella, “Pro-drugs as NovelDelivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987.

For example, if the compound of the invention contains a carboxylic acidfunctional group, a prodrug can comprise an ester formed by thereplacement of the hydrogen atom of the acid group with a group such as(C₁-C₈)alkyl, (C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl havingfrom 4 to 9 Carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5to 10 Carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 Carbonatoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 Carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 Carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 Carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 Carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N-(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

Similarly, if the compound of the present invention comprises an alcoholfunctional group, a prodrug can be formed by the replacement of thehydrogen atom of the alcohol group with a group such as(C₁-C₆)alkanoyloxymethyl, 1-((C₁-C₆)alkanoyloxy)ethyl,1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl, (C₁-C₆)alkoxycarbonyloxymethyl,N-(C₁-C₆)alkoxycarbonylaminomethyl, succinoyl, (C₁-C₆)alkanoyl,α-amino(C₁-C₄)alkanoyl, arylacyl and α-aminoacyl, orα-aminoacyl-α-aminoacyl, where each α-aminoacyl group is independentlyselected from the naturally occurring L-amino acids, P(O)(OH)₂,—P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radical resulting from theremoval of a hydroxyl group of the hemiacetal form of a carbohydrate).

If the compound of the present invention comprises an amine functionalgroup, a prodrug can be formed by the replacement of a hydrogen atom inthe amine group with a group such as R-carbonyl, RO-carbonyl,NRR′-carbonyl where R and R′ are each independently ((C₁-C₁₀)alkyl,(C₃-C₇)cycloalkyl, benzyl, or R-carbonyl is a natural α-aminoacyl ornatural α-aminoacyl-natural α-aminoacyl, —C(OH)C(O)OY wherein (Y is H,(C₁-C₆)alkyl or benzyl), —C(OY₀)Y₁ wherein Y₀ is (C₁-C₄) alkyl and Y₁ is((C₁-C₆)alkyl, carboxy(C₁-C₆)alkyl, amino(C₁-C₄)alkyl or mono-N— ordi-N,N-(C₁-C₆)alkylaminoalkyl, —C(Y₂)Y₃ wherein Y₂ is H or methyl and Y₃is mono-N— or di-N,N-(C₁-C₆)alkylamino, morpholino, piperidin-1-yl orpyrrolidin-1-yl.

The compounds of the present invention may contain asymmetric or chiralcenters, and therefore, exist in different stereoisomeric forms. It iscontemplated that all stereoisomeric forms of the compounds as well asmixtures thereof, including racemic mixtures, form part of the presentinvention. In addition, the present invention contemplates all geometricand positional isomers. For example, if the compound contains a doublebond, both the cis and trans forms, as well as mixtures, arecontemplated.

Diasteromeric mixtures can be separated into their individualstereochemical components on the basis of their physical chemicaldifferences by methods known per se, for example, by chromatographyand/or fractional crystallization. Enantiomers can be separated byconverting the enantiomeric mixture into a diasteromeric mixture byreaction with an appropriate optically active compound (e.g., alcohol),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereomers to the corresponding pure enantiomers. Also,some of the compounds of this invention may be atropisomers (e.g.,substituted biaryls) and are considered as part of this invention.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like. The present invention contemplates andencompasses both the solvated and unsolvated forms.

It is also possible that compounds of the present invention may exist indifferent tautomeric forms. All tautomers of compounds of the presentinvention are contemplated. For example, all of the tautomeric forms ofthe imidazole moiety are included in this invention. Also, for example,all keto-enol or imine-enamine forms of the compounds are included inthis invention.

Those skilled in the art will recognize that the compound namescontained herein may be based on a particular tautomer of a compound.While the name for only a particular tautomer may be used, it isintended that all tautomers are encompassed by the name of theparticular tautomer and included as part of the invention.

It is also intended that the invention disclosed herein encompasscompounds that are synthesized in vitro using laboratory techniques,such as those well known to synthetic chemists; or synthesized using invivo techniques, such as through metabolism, fermentation, digestion,and the like. It is also contemplated that the compounds of the presentinvention may be synthesized using a combination of in vitro and in vivotechniques.

The present invention also includes isotopically-labelled compounds,which are identical to those recited herein, but for the fact that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁸Cl, respectively. Compounds of thepresent invention that contain the aforementioned isotopes and/or otherisotopes of other atoms are within the scope of this invention. Certainisotopically-labelled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detection.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labelled compounds of Formula I of this invention andprodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the Schemes and/or in the Examples below, bysubstituting a readily available isotopically labelled reagent for anon-isotopically labelled reagent.

In general the compounds of this invention can be made by processeswhich include processes analogous to those known in the chemical arts,particularly in light of the description contained herein.

In another aspect, the present invention concerns the treatment ofdiabetes, including impaired glucose tolerance, insulin resistance,insulin dependent diabetes mellitus (Type I) and non-insulin dependentdiabetes mellitus (NIDDM or Type II). Also included in the treatment ofdiabetes are the treatment of the diabetic complications, such asneuropathy, nephropathy, retinopathy or cataracts.

Diabetes can be treated by administering to a patient having diabetes(Type I or Type II), insulin resistance, impaired glucose tolerance, orany of the diabetic complications such as neuropathy, nephropathy,retinopathy or cataracts, a therapeutically effective amount of acompound of the present invention. It is also contemplated that diabetesbe treated by administering a compound of the present invention or another glycogen phosphorylase inhibitor in combination with an additionalagent that can be used to treat diabetes and/or obesity. Preferredgylcogen phosphorylase inhibitors that are useful in combination withother agents useful to treat diabetes and/or obesity include those ofFormula I. Additional preferred gylcogen phosphorylase inhibitors aredisclsoed in PCT publications WO 96/39384 and WO 96/39385.

Representative agents that can be used to treat diabetes include insulinand insulin analogs: (e.g., LysPro insulin inhaled formulationscomprising insulin); GLP-1 (7-37) (insulinotropin) and GLP-1 (7-36)-NH₂;sulfonylureas and analogs: chlorpropamide, glibenclamide, tolbutamide,tolazamide, acetohexamide, glypizide, glimepiride, repaglinide,meglitinide; biguanides: metformin, phenformin, buformin; α2-antagonistsand imidazolines: midaglizole, isaglidole, deriglidole, idazoxan,efaroxan, fluparoxan; other insulin secretagogues: linogliride,insulinotropin, exendin-4, BTS-67582, A-4166; glitazones: ciglitazone,pioglitazone, englitazone, troglitazone, darglitazone, rosiglitazone;PPAR-gamma agonists; RXR agonists: JTT-501, MCC-555, MX-6054,DRF2593,GI-262570, KRP-297, LG100268; fatty acid oxidation inhibitors:clomoxir, etomoxir; α-glucosidase inhibitors: precose, acarbose,miglitol, emiglitate, voglibose, MDL-25,637, camiglibose, MDL-73,945;β-agonists: BRL 35135, BRL 37344, Ro 16-8714, ICI D7114, CL 316,243,TAK-667, AZ40140; phosphodiesterase inhibitors, both cAMP and cGMP type:sildenafil, L686398: L-386,398; lipid-lowering agents: benfluorex,atorvastatin; antiobesity agents: fenfluramine, orlistat, sibutramine;vanadate and vanadium complexes (e.g., Naglivan®) and peroxovanadiumcomplexes; amylin antagonists: pramlintide, AC-137; lipoxygenaseinhibitors: masoprocal; somatostatin analogs: BM-23014, seglitide,octreotide; glucagon antagonists: BAY 276-9955; insulin signalingagonists, insulin mimetics, PTP1B inhibitors: L-783281, TER17411,TER17529; gluconeogenesis inhibitors: GP3034; somatostatin analogs andantagonists; antilipolytic agents: nicotinic acid, acipimox, WAG 994;glucose transport stimulating agents: BM-130795; glucose synthase kinaseinhibitors: lithium chloride, CT98014, CT98023; galanin receptoragonisnts; MTP inhibitors such as those disclosed in U.S. provisionalpatent application No. 60/164,803; growth hormone secretagogues such asthose disclosed in PCT publication numbers WO 97/24369 and WO 98/58947;NPY antagonists: PD-160170, BW-383, BW1229, CGP-71683A, NGD 95-1,L-152804; Anorectic agents inlcuding 5-HT and 5-HT2C receptorantagonists. and/or mimetics: dexfenfluramine, Prozac®, Zoloft®; CCKreceptor agonists: SR-27897B; galanin receptor antagonists; MCR-4antagonists: HP-228; leptin or mimetics:leptin; 11-beta-hydroxysteroiddehydrogenase type-I inhibitors; urocortin mimetics, CRF antagonists,and CRF binding proteins: RU-486, urocortin. Other anti-diabetic agentsthat can be used in combination with a glycogen phosphorylase inhibitorinclude ergoset and D-chiroinositol. Any combination of agents can beadministered as described above.

In addition to the categories and compounds mentioned above, gylcogenphosphorylase inhibitors, preferrably the compounds of the presentinvention, can be administered in combination with thyromimeticcompounds, aldose reductase inhibitors, glucocorticoid receptorantagonists, NHE-1 inhibitors, or sorbitol dehydrogenase inhibitors, orcombinations thereof, to treat or prevent diabetes, insulin resistance,diabetic neuropathy, diabetic nephropathy, diabetic retinopathy,cataracts, hyperglycemia, hypercholesterolemia, hypertension,hyperinsulinemia, hyperlipidemia, atherosclerosis, or tissue ischemia,particularly myocardial ischemia.

It is generally accepted that thyroid hormones, specifically,biologically active iodothyronines, are critical to normal developmentand to maintaining metabolic homeostasis. Thyroid hormones stimulate themetabolism of cholesterol to bile acids and enhance the lipolyticresponses of fat cells to other hormones. U.S. Pat. Nos. 4,766,121;4,826,876; 4,910,305; and 5,061,798 disclose certain thyroid hormonemimetics (thyromimetics), namely,3,5-dibromo-3′-[6-oxo-3(1H)-pyridazinylmethyl]-thyronines. U.S. Pat. No.5,284,971 discloses certain thyromimetic cholesterol lowering agents,namely, 4-(3-Cyclohexyl-4-hydroxy or -methoxy phenylsulfonyl)-3,5dibromo-phenylacetic compounds. U.S. Pat. Nos. 5,401,772; 5,654,468; and5,569,674 disclose certain thyromimetics that are lipid lowering agents,namely, heteroacetic acid derivatives. In addition, certain oxamic acidderivatives of thyroid hormones are known in the art. For example, N.Yokoyama, et al. in an article published in the Journal of MedicinalChemistry, 38 (4): 695-707 (1995) describe replacing a —CH₂ group in anaturally occurring metabolite of T₃ with an —NH group resulting in—HNCOCO₂H. Likewise, R. E. Steele et al. in an article published inInternational Congressional Service (Atherosclerosis X) 1066: 321-324(1995) and Z. F. Stephan et al. in an article published inAtherosclerosis, 126: 53-63 (1996), describe certain oxamic acidderivatives useful as lipid-lowering thyromimetic agents, yet devoid ofundesirable cardiac activities. Other useful thyromimetics that can beused in combination with a glycogen phosphorylase inhibitor includeCGS-26214.

Each of the thyromimetic compounds referenced above and otherthyromimetic compounds can be used in combination with the compounds ofthe present invention to treat or prevent diabetes, insulin resistance,diabetic neuropathy, diabetic nephropathy, diabetic retinopathy,cataracts hyperglycemia, hypercholesterolemia, hypertension,hyperinsulinemia, hyperlipidemia, atherosclerosis, or tissue ischemia.

The compounds of the present invention can also be used in combinationwith aldose reductase inhibitors. Aldose reductase inhibitors constitutea class of compounds that have become widely known for their utility inpreventing and treating conditions arising from complications ofdiabetes, such as diabetic neuropathy and nephropathy. Such compoundsare well known to those skilled in the art and are readily identified bystandard biological tests. For example, the aldose reductase inhibitorszopolrestat, 1-phthalazineacetic acid,3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl]methyl]-, andrelated compounds are described in U.S. Pat. No. 4,939,140 to Larson etal.

Aldose reductase inhibitors have been taught for use in lowering lipidlevels in mammals. See, for example, U.S. Pat. No. 4,492,706 toKallai-sanfacon and EP 0 310 931 A2 (Ethyl Corporation).

U.S. Pat. No. 5,064,830 to Going discloses the use of certainoxophthalazinyl acetic acid aldose reductase inhibitors, includingzopolrestat, for lowering of blood uric acid levels.

Commonly assigned U.S. Pat. No. 5,391,551 discloses the use of certainaldose reductase inhibitors, including zopolrestat, for lowering bloodlipid levels in humans. The disclosure teaches that therapeuticutilities derive from the treatment of diseases caused by an increasedlevel of triglycerides in the blood, such diseases includecardiovascular disorders such as thrombosis, arteriosclerosis,myocardial infarction, and angina pectoris. A preferred aldose reductaseinhibitor is 1-phthalazineacetic acid,3,4-dihydro-4-oxo-3-[[5-trifluoromethyl)-2-benzothiazolyl]methyl]-, alsoknown as zopolrestat.

The term aldose reductase inhibitor refers to compounds that inhibit thebioconversion of glucose to sorbitol, which is catalyzed by the enzymealdose reductase.

Any aldose reductase inhibitor may be used in a combination with acompound of the present invention. Aldose reductase inhibition isreadily determined by those skilled in the art according to standardassays (J. Malone, Diabetes, 29:861-864 (1980). “Red Cell Sorbitol, anIndicator of Diabetic Control”). A variety of aldose reductaseinhibitors are described herein; however, other aldose reductaseinhibitors useful in the compositions and methods of this invention willbe known to those skilled in the art.

The activity of an aldose reductase inhibitor in a tissue can bedetermined by testing the amount of aldose reductase inhibitor that isrequired to lower tissue sorbitol (i.e., by inhibiting the furtherproduction of sorbitol consequent to blocking aldose reductase) or lowertissue fructose (by inhibiting the production of sorbitol consequent toblocking aldose reductase and consequently the production of fructose.

Accordingly, examples of aldose reductase inhibitors useful in thecompositions, combinations and methods of the present invention include:

1. 3-(4-bromo-2-fluorobenzyl)-3,4-dihydro-4-oxo-1-phthalazineacetic acid(ponalrestat, U.S. Pat. No. 4,251,528);

2.N[[(5-trifluoromethyl)-6-methoxy-1-naphthalenyl]thioxomethyl]-N-methylglycine(tolrestat, U.S. Pat. No. 4,600,724);

3. 5-[(Z,E)-β-methylcinnamylidene]-4-oxo-2-thioxo-3-thiazolideneaceticacid (epalrestat, U.S. Pat. No. 4,464,382, U.S. Pat. No. 4,791,126, U.S.Pat. No. 4,831,045);

4.3-(4-bromo-2-fluorobenzyl)-7-chloro-3,4-dihydro-2,4-dioxo-1(2H)-quinazolineaceticacid (zenarestat, U.S. Pat. Nos. 4,734,419, and 4,883,800);

5. 2R,4R-6,7-dichloro-4-hydroxy-2-methylchroman-4-acetic acid (U.S. Pat.No. 4,883,410);

6. 2R,4R-6,7-dichloro-6-fluoro-4-hydroxy-2-methylchroman-4-acetic acid(U.S. Pat. No. 4,883,410);

7. 3,4-dihydro-2,8-diisopropyl-3-oxo-2H-1,4-benzoxazine-4-acetic acid(U.S. Pat. No. 4,771,050);

8.3,4-dihydro-3-oxo-4-[(4,5,7-trifluoro-2-benzothiazolyl)methyl]-2H-1,4-benzothiazine-2-aceticacid (SPR-210, U.S. Pat. No. 5,252,572);

9.N-[3,5-dimethyl-4-[(nitromethyl)sulfonyl]phenyl]-2-methyl-benzeneacetamide(ZD5522, U.S. Pat. No. 5,270,342 and U.S. Pat. No. 5,430,060);

10. (S)-6-fluorospiro[chroman-4,4′-imidazolidine]-2,5′-dione (sorbinil,U.S. Pat. No. 4,130,714);

11. d-2-methyl-6-fluoro-spiro(chroman-4′,4′-imidazolidine)-2′,5′-dione(U.S. Pat. No. 4,540,704);

12. 2-fluoro-spiro(9H-fluorene-9,4′-imidazolidine)2′,5′-dione (U.S. Pat.No. 4,438,272);

13. 2,7-di-fluoro-spiro(9H-fluorene-9,4′-imidazolidine)2′,5′-dione (U.S.Pat. No. 4,436,745, U.S. Pat. No. 4,438,272);

14.2,7-di-fluoro-5-methoxy-spiro(9H-fluorene-9,4′-imidazolidine)2′,5′-dione(U.S. Pat. No. 4,436,745, U.S. Pat. No. 4,438,272);

15. 7-fluoro-spiro(5H-indenol[1,2-b]pyridine-5,3′-pyrrolidine)2,5′-dione(U.S. Pat. No. 4,436,745, U.S. Pat. No. 4,438,272);

16.d-cis-6′-chloro-2′,3′-dihydro-2′-methyl-spiro-(imidazolidine-4,4′-4′-H-pyrano(2,3-b)pyridine)-2,5-dione(U.S. Pat. No. 4,980,357);

17.spiro[imidazolidine-4,5′(6H)-quinoline]2,5-dione-3′-chloro-7,′8′-dihydro-7′-methyl-(5′-cis)(U.S.Pat. No. 5,066,659);

18.(2S,4S)-6-fluoro-2′,5′-dioxospiro(chroman-4,4′-imidazolidine)-2-carboxamide(U.S. Pat. No. 5,447,946); and

19.2-[(4-bromo-2-fluorophenyl)methyl]-6-fluorospiro[isoquinoline-4(1H),3′-pyrrolidine]-1,2′,3,5′(2H)-tetrone(ARI-509, U.S. Pat. No. 5,037,831).

Other aldose reductase inhibitors include compounds having formula Iabelow

or a pharmaceutically acceptable salt or prodrug thereof, wherein

Z is O or S;

R¹ is hydroxy or a group capable of being removed in vivo to produce acompound of formula I wherein R¹ is OH; and

X and Y are the same or different and are selected from hydrogen,trifluoromethyl, fluoro, and chloro.

A preferred subgroup within the above group of aldose reductaseinhibitors includes numbered compounds 1, 2, 3, 4, 5, 6, 9, 10, and 17,and the following compounds of Formula Ia:

20.3,4-dihydro-3-(5-fluorobenzothiazol-2-ylmethyl)-4-oxophthalazin-1-yl-aceticacid [R¹=hydroxy; X═F; Y═H];

21.3-(5,7-difluorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X═Y═F];

22.3-(5-chlorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X═Cl; Y═H];

23.3-(5,7-dichlorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X═Y═Cl];

24.3,4-dihydro-4-oxo-3-(5-trifluoromethylbenzoxazol-2-ylmethyl)phthalazin-1-ylaceticacid [R¹=hydroxy; X═CF₃; Y═H];

25.3,4-dihydro-3-(5-fluorobenzoxazol-2-ylmethyl)-4-oxophthalazin-1-yl-aceticacid [R¹=hydroxy; X═F; Y═H];

26.3-(5,7-difluorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X═Y═F];

27.3-(5-chlorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X═Cl; Y═H];

28.3-(5,7-dichlorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X═Y═Cl]; and

29. zopolrestat; 1-phthalazineacetic acid,3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl]methyl]-[R¹=hydroxy;X=trifluoromethyl; Y═H].

In compounds 20-23, and 29 Z is S. In compounds 24-28, Z is O.

Of the above subgroup, compounds 20-29 are more preferred with 29especially preferred. Procedures for making the aldose reducataseinhibitors of formula Ia can be found in PCT publication number WO99/26659.

Each of the aldose reductase inhibitors referenced above and otheraldose reductase inhibitors can be used in combination with thecompounds of the present invention to treat diabetes, insulinresistance, diabetic neuropathy, diabetic nephropathy, diabeticretinopathy, cataracts, hyperglycemia, hypercholesterolemia,hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, ortissue ischemia.

The compounds of the present invention can also be used in combinationwith glucocorticoid receptor antagonists. The glucocorticoid receptor(GR) is present in glucocorticoid responsive cells where it resides inthe cytosol in an inactive state until it is stimulated by an agonist.Upon stimulation the glucocorticoid receptor translocates to the cellnucleus where it specifically interacts with DNA and/or protein(s) andregulates transcription in a glucocorticoid responsive manner. Twoexamples of proteins that interact with the glucocorticoid receptor arethe transcription factors, API and NFκ-B. Such interactions result ininhibition of API- and NFκ-B-mediated transcription and are believed tobe responsible for the anti-inflammatory activity of endogenouslyadministered glucocorticoids. In addition, glucocorticoids may alsoexert physiologic effects independent of nuclear transcription.Biologically relevant glucocorticoid receptor agonists include cortisoland corticosterone. Many synthetic glucocorticoid receptor agonistsexist including dexamethasone, prednisone and prednisilone. Bydefinition, glucocorticoid receptor antagonists bind to the receptor andprevent glucocorticoid receptor agonists from binding and eliciting GRmediated events, including transcription. RU486 is an example of anon-selective glucocorticoid receptor antagonist. GR antagonists can beused in the treatment of diseases associated with an excess or adeficiency of glucocorticoids in the body. As such, they may be used totreat the following: obesity, diabetes, cardiovascular disease,hypertension, Syndrome X, depression, anxiety, glaucoma, humanimmunodeficiency virus (HIV) or acquired immunodeficiency syndrome(AIDS), neurodegeneration (for example, Alzheimer's and Parkinson's),cognition enhancement, Cushing's Syndrome, Addison's Disease,osteoporosis, frailty, inflammatory diseases (such as osteoarthritis,rheumatoid arthritis, asthma and rhinitis), tests of adrenal function,viral infection, immunodeficiency, immunomodulation, autoimmunediseases, allergies, wound healing, compulsive behavior, multi-drugresistance, addiction, psychosis, anorexia, cachexia, post-traumaticstress syndrome, post-surgical bone fracture, medical catabolism andprevention of muscle frailty. Examples or GR antagonists that can beused in combination with a compound of the present invention includecompounds of formula Ib below:

an isomer thereof, a prodrug of said compound or isomer, or apharmaceutically acceptable salt of said compound, isomer or prodrug;wherein m is 1 or 2;

- - - represents an optional bond;

A is selected from the group consisting of

D is CR₇, CR₇R₁₆, N, NR₇ or O;

E is C, CR₆ or N;

F is CR₄, CR₄R₅ or O;

G, H and I together with 2 Carbon atoms from the A-ring or 2 Carbonatoms from the B-ring form a 5-membered heterocyclic ring comprising oneor more N, O or S atoms; provided that there is at most one of O and Sper ring; J, K, L and M together with 2 Carbon atoms from the B-ringforms a 6-membered heterocyclic ring comprising 1 or more N atoms;

X is a) absent, b) —CH₂—, c) —CH(OH)— or d) —C(O)—;

R₁ is a) —H, b) -Z-CF₃, c) —(C₁-C₆)alkyl, d) —(C₂-C₆)alkenyl, e)—(C₂-C₆)alkynyl, f) —CHO, g) —CH═N—OR₁₂, h) -Z-C(O)OR₁₂, i)-Z-C(O)—NR₁₂R₁₃, j) -Z-C(O)—NR₁₂-Z-het, k) -Z-NR₁₂R₁₃, l) -Z-NR₁₂het, m)-Z-het, n) -Z-O-het, o) -Z-aryl′, p) -Z-O-aryl′, q) —CHOH-aryl′ or r)—C(O)-aryl′ wherein aryl′ in substituents o) to r) is substitutedindependently with 0, 1 or 2 of the following: -Z-OH, -Z-NR₁₂R₁₃,-Z-NR₁₂-het, —C(O)NR₁₂R₁₃, —C(O)O(C₁-C₆)alkyl, —C(O)OH, —C(O)-het,—NR₁₂—C(O)—(C₁-C₆)alkyl, —NR₁₂—C(O)—(C₂-C₆)alkenyl,—NR₁₂—C(O)—(C₂-C₆)alkynyl, —NR₁₂—C(O)-Z-het, —CN, -Z-het,—O—(C₁-C₃)alkyl-C(O)—NR₁₂R₁₃, —O—(C₁-C₃)alkyl-C(O)O(C₁-C₆)alkyl,—NR₁₂-Z-C(O)O(C₁-C₆)alkyl, —N(Z-C(O)O(C₁-C₆)alkyl)₂,—NR₁₂-Z-C(O)—NR₁₂R₁₃, -Z-NR₁₂—SO₂—R₁₃, —NR₁₂—SO₂-het, —C(O)H,-Z-NR₁₂-Z-O(C₁-C₆)alkyl, -Z-NR₁₂-Z-NR₁₂R₁₃, -Z-NR₁₂—(C₃-C₆)cycloalkyl,-Z-N(Z-O(C₁-C₆)alkyl)₂, —SO₂R₁₂, —SOR₁₂, —SR₁₂, —SO₂NR₁₂R₁₃,—O—C(O)—(C₁-C₄)alkyl, —O—SO₂—(C₁-C₄)alkyl, -halo or —CF₃;

Z for each occurrence is independently a) —(C₀-C₆)alkyl, b)—(C₂-C₆)alkenyl or c) —(C₂-C₆)alkynyl;

R₂ is a) —H, b) -halo, c) —OH, d) —(C₁-C₆)alkyl substituted with 0 or 1—OH, e) —NR₁₂R₁₃, f) -Z-C(O)O(C₁-C₆)alkyl, g) -Z-C(O)NR₁₂R₁₃, h)—O—(C₁-C₆)alkyl, i) -Z-O—C(O)—(C₁-C₆)alkyl, j)-Z-O—(C₁-C₃)alkyl-C(O)—NR₁₂R₁₃, k) -Z-O—(C₁-C₃)alkyl-C(O)—O(C₁-C₆)alkyl,l) —O—(C₂-C₆)alkenyl, m) —O—(C₂-C₆)alkynyl, n) —O-Z-het, o) —COOH, p)—C(OH)R₁₂R₁₃ or q) -Z-CN;

R₃ is a) —H, b) —(C₁-C₁₀)alkyl wherein 1 or 2 Carbon atoms, other thanthe connecting carbon atom, may optionally be replaced with 1 or 2heteroatoms independently selected from S, O and N and wherein eachcarbon atom is substituted with 0, 1 or 2 R_(y), c) —(C₂-C₁₀)alkenylsubstituted with 0, 1 or 2 R_(y), d) —(C₂-C₁₀)alkynyl wherein 1 Carbonatom, other than the connecting carbon atom, may optionally be replacedwith 1 oxygen atom and wherein each carbon atom is substituted with 0, 1or 2 R_(y), e) —CH═C═CH₂, f) —CN, g) —(C₃-C₆)cycloalkyl, h) -Z-aryl, i)-Z-het, j) —C(O)O(C₁-C₆)alkyl, k) —O(C₁-C₆)alkyl, I) -Z-S—R₁₂, m)-Z-S(O)—R₁₂, n) -Z-S(O)₂—R₁₂, o) —CF₃ p) —NR₁₂O—(C₁-C₆)alkyl or q)—CH₂OR_(y);

provided that one of R₂ and R₃ is absent when there is a double bondbetween CR₂R₃ (the 7 position) and the F moiety (the 8 position) of theC-ring;

R_(y) for each occurrence is independently a) —OH, b) -halo, c) -Z-CF₃,d) -Z-CF(C₁-C₃ alkyl)₂, e) —CN, f) —NR₁₂R₁₃, g) —(C₃-C₆)cycloalkyl, h)—(C₃-C₆)cycloalkenyl, i) —(C₀-C₃)alkyl-aryl, j) -het or k) —N₃;

or R₂ and R₃ are taken together to form a) ═CHR₁₁, b) ═NOR₁₁, c) ═O, d)═N—NR₁₂, e) ═N—NR₁₂—C(O)—R₁₂, f) oxiranyl or g) 1,3-dioxolan-4-yl;

R₄ and R₅ for each occurrence are independently a) —H, b) —CN, c)—(C₁-C₆)alkyl substituted with 0 to 3 halo, d) —(C₂-C₆)alkenylsubstituted with 0 to 3 halo, e) —(C₂-C₆)alkynyl substituted with 0 to 3halo, f) —O—(C₁-C₆)alkyl substituted with 0 to 3 halo, g)—O—(C₂-C₆)alkenyl substituted with 0 to 3 halo, h) —O—(C₂-C₆)alkynylsubstituted with 0 to 3 halo, i) halo, j) —OH, k) (C₃-C₆)cycloalkylor 1) (C₃-C₆)cycloalkenyl;

or R₄ and R₅ are taken together to form ═O;

R₆ is a) —H, b) —CN, c) —(C₁-C₆)alkyl substituted with 0 to 3 halo, d)—(C₂-C₆)alkenyl substituted with 0 to 3 halo, e) —(C₂-C₆)alkynylsubstituted with 0 to 3 halo or f) —OH;

R₇ and R₁₆ for each occurrence are independently a) —H, b) -halo, c)—CN, d) —(C₁-C₆)alkyl substituted with 0 to 3 halo, e) —(C₂-C₆)alkenylsubstituted with 0 to 3 halo or f) —(C₂-C₆)alkynyl substituted with 0 to3 halo; provided that R₇ is other than —CN or -halo when D is NR₇;

or R₇ and R₁₆ are taken together to form ═O;

R₈, R₉, R₁₄ and R₁₅ for each occurrence are independently a) —H, b)-halo, c) (C₁-C₆)alkyl substituted with 0 to 3 halo, d) —(C₂-C₆)alkenylsubstituted with 0 to 3 halo, e) —(C₂-C₆)alkynyl substituted with 0 to 3halo, f) —CN, g) —(C₃-C₆)cycloalkyl, h) —(C₃-C₆)cycloalkenyl, i) —OH, j)—O—(C₁-C₆)alkyl, k) —O—(C₁-C₆)alkenyl, l) —O—(C₁-C₈)alkynyl, m)—NR₁₂R₁₃, n) —C(O)OR₁₂ or o) —C(O)NR₁₂R₁₃;

or R₈ and R₉ are taken together on the C-ring to form ═O; provided thatwhen m is 2, only one set of R₈ and R⁹ are taken together to form ═O;

or R₁₄ and R₁₅ are taken together to form ═O; provided that when R₁₄ andR₁₅ are taken together to form ═O, D is other than CR₇ and E is otherthan C;

R₁₀ is a) —(C₁-C₁₀)alkyl substituted with 0 to 3 substituentsindependently selected from -halo, —OH and —N₃, b) —(C₂-C₁₀)alkenylsubstituted with 0 to 3 substituents independently selected from -halo,—OH and —N₃, c) —(C₂-C₁₀)alkynyl substituted with 0 to 3 substituentsindependently selected from -halo, —OH and —N₃, d) -halo, e) -Z-CN, f)—OH, g) -Z-het, h) -Z-NR₁₂R₁₃, i) -Z-C(O)-het, j) -Z-C(O)—(C₁-C₆)alkyl,k) -Z-C(O)—NR₁₂R₁₃, l) -Z-C(O)—NR₁₂-Z-CN, m) -Z-C(O)—NR₁₂-Z-het, n)-Z-C(O)—NR₁₂-Z-aryl, o) -Z-C(O)—NR₁₂-Z-NR₁₂R₁₃, p)-Z-C(O)—NR₁₂-Z-O(C₁-C₆)alkyl, q) —(C₁-C₆)alkyl-C(O)OH, r)-Z-C(O)O(C₁-C₆)alkyl, s) -Z-O—(C₀-C₆)alkyl-het, t)-Z-O—(C₀-C₆)alkyl-aryl, u) -Z-O—(C₁-C₆)alkyl substituted with 0 to 2R_(x), v) -Z-O—(C₁-C₆)alkyl-CH(O), w) -Z-O—(C₁-C₆)alkyl-NR₁₂-het, x)-Z-O-Z-het-Z-het, y) -Z-O-Z-het-Z-NR₁₂R₁₃, z) -Z-O-Z-het-C(O)-het, a1)-Z-O-Z-C(O)-het, b1) -Z-O-Z-C(O)-het-het, c1) -Z-O-Z-C(O)—(C₁-C₆)alkyl,d1) -Z-O-Z-C(S)—NR₁₂R₁₃, e1) -Z-O-Z-C(O)—NR₁₂R₁₃, f1)-Z-O-Z-(C₁-C₃)alkyl-C(O)—NR₁₂R₁₃, g1) -Z-O-Z-C(O)—O(C₁-C₆)alkyl, h1)-Z-O-Z-C(O)—OH, i1) -Z-O-Z-C(O)—NR₁₂—O(C₁-C₆)alkyl, j1)-Z-O-Z-C(O)—NR₁₂—OH, k1) -Z-O-Z-C(O)—NR₁₂-Z-NR₁₂R₁₃, l1)-Z-O-Z-C(O)—NR₁₂-Z-het, m1) -Z-O-Z-C(O)—NR₁₂—SO₂—(C₁-C₆)alkyl, n1)-Z-O-Z-C(═NR₁₂)(NR₁₂R₁₃), o1) -Z-O-Z-C(═NOR₁₂)(NR₁₂R₁₃), p1)-Z-NR₁₂—C(O)—O-Z-NR₁₂R₁₃, q1) -Z-S—C(O)—NR₁₂R₁₃, r1)-Z-O—SO₂—(C₁-C₆)alkyl, s1) -Z-O—SO₂-aryl, t1) -Z-O—SO₂—NR₁₂R₁₃, u1)-Z-O—SO₂—CF₃, v1) -Z-NR₁₂C(O)OR₁₃ or w1) -Z-NR₁₂C(O)R₁₃;

or R₉ and R₁₀ are taken together on the moiety of formula A-5 to form a)═O or b) ═NOR₁₂;

R₁₁ is a) —H, b) —(C₁-C₅)alkyl, c) —(C₃-C₆)cycloalkyl or d)—(C₀-C₃)alkyl-aryl;

R₁₂ and R₁₃ for each occurrence are each independently a) —H, b)—(C₁-C₆)alkyl wherein 1 or 2 Carbon atoms, other than the connectingcarbon atom, may optionally be replaced with 1 or 2 heteroatomsindependently selected from S, O and N and wherein each carbon atom issubstituted with 0 to 6 halo, c) —(C₂-C₆)alkenyl substituted with 0 to 6halo or d) —(C₁-C₆)alkynyl wherein 1 Carbon atom, other than theconnecting carbon atom, may optionally be replaced with 1 oxygen atomand wherein each carbon atom is substituted with 0 to 6 halo;

or R₁₂ and R₁₃ are taken together with N to form het;

or R₆ and R₁₄ or R₁₅ are taken together to form 1,3-dioxolanyl;

aryl is a) phenyl substituted with 0 to 3 R_(x), b) naphthyl substitutedwith 0 to 3 R_(x) or c) biphenyl substituted with 0 to 3 R_(x);

het is a 5-,6- or 7-membered saturated, partially saturated orunsaturated ring containing from one (1) to three (3) heteroatomsindependently selected from the group consisting of nitrogen, oxygen andsulfur; and including any bicyclic group in which any of the aboveheterocyclic rings is fused to a benzene ring or another heterocycle;and the nitrogen may be in the oxidized state giving the N-oxide form;and substituted with 0 to 3 R_(x);

R_(x) for each occurrence is independently a) -halo, b) —OH, c)—(C₁-C₆)alkyl, d) —(C₂-C₆)alkenyl, e) —(C₂-C₆)alkynyl, f)—O(C₁-C₆)alkyl, g) —O(C₂-C₆)alkenyl, h) —O(C₂-C₆)alkynyl, i)—(C₀-C₆)alkyl-NR₁₂R₁₃, j) —C(O)—NR₁₂R₁₃, k) -Z-SO₂R₁₂, l) -Z-SOR₁₂, m)-Z-SR₁₂, n) —NR₁₂—SO₂R₁₃, o) —NR₁₂—C(O)—R₁₃, p) —NR₁₂—OR₁₃, q)—SO₂—NR₁₂R₁₃, r) —CN, s) —CF₃, t) —C(O)(C₁-C₆)alkyl, u)=O, v)-Z-SO₂-phenyl or w) -Z-SO₂-het′;

aryl′ is phenyl, naphthyl or biphenyl;

het′ is a 5-,6- or 7-membered saturated, partially saturated orunsaturated ring containing from one (1) to three (3) heteroatomsindependently selected from the group consisting of nitrogen, oxygen andsulfur; and including any bicyclic group in which any of the aboveheterocyclic rings is fused to a benzene ring or another heterocycle;

provided that:

1) X—R₁ is other than hydrogen or methyl;

2) when R₉ and R₁₀ are substituents on the A-ring, they are other thanmono- or di-methoxy;

3) when R₂ and R₃ are taken together to form ═CHR₁₁ or ═O wherein R₁₁ is—O(C₁-C₆)alkyl, then —X—R₁ is other than (C₁-C₄)alkyl;

4) when R₂ and R₃ taken together are C═O and R₉ is hydrogen on theA-ring; or when R₂ is hydroxy, R₃ is hydrogen and R₉ is hydrogen on theA-ring, then R₁₀ is other than —O—(C₁-C₆)alkyl or —O—CH₂-phenyl at the2-position of the A-ring;

5) when X—R₁ is (C₁-C₄)alkyl, (C₂-C₄)alkenyl or (C₂-C₄)alkynyl, R₉ andR₁₀ are other than mono-hydroxy or ═O, including the diol form thereof,when taken together; and

6) when X is absent, R₁ is other than a moiety containing a heteroatomindependently selected from N, O or S directly attached to the junctureof the B-ring and the C-ring. (See U.S. Provisional Patent ApplicationNo. 60/132,130)

Each of the glucocorticoid receptor antagonists referenced above andother glucocorticoid receptor antagonists can be used in combinationwith the compounds of the present invention to treat or preventdiabetes, hyperglycemia, hypercholesterolemia, hypertension,hyperinsulinemia, hyperlipidemia, atherosclerosis, or tissue ischemia.

The compounds of the present invention can also be used in combinationwith sorbitol dehydrogenase inhibitors. Sorbitol dehydrogenaseinhibitors lower fructose levels and have been used to treat or preventdiabetic complications such as neuropathy, retinopathy, nephropathy,cardiomyopathy, microangiopathy, and macroangiopathy. U.S. Pat. Nos.5,728,704 and 5,866,578 disclose compounds and a method for treating orpreventing diabetic complications by inhibiting the enzyme sorbitoldehydrogenase.

Each of the sorbitol dehydrogenase inhibitors referenced above and othersorbitol dehydrogenase inhibitors can be used in combination with thecompounds of the present invention to treat diabetes, insulinresistance, diabetic neuropathy, diabetic nephropathy, diabeticretinopathy, cataracts, hyperglycemia, hypercholesterolemia,hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis, ortissue ischemia.

The compounds of the present invention can also be used in combinationwith sodium-hydrogen exchanger type 1 (NHE-1) inhibitors. NHE-1inhibitors can be used to reduce tissue damage resulting from ischemia.Of great concern is tissue damage that occurs as a result of ischemia incardiac, brain, liver, kidney, lung, gut, skeletal muscle, spleen,pancreas, nerve, spinal cord, retina tissue, the vasculature, orintestinal tissue. NHE-1 inhibitors can also be administered to preventperioperative myocardial ischemic injury.

Examples of NHE-1 inhibitors include a compound having the Formula Ic

Formula Ic

a prodrug thereof or a pharmaceutically acceptable salt of said compoundor of said prodrug, wherein

Z is carbon connected and is a five-membered, diaza, diunsaturated ringhaving two contiguous nitrogens, said ring optionally mono-, di-, ortri-substituted with up to three substituents independently selectedfrom R¹, R² and R³; or

Z is carbon connected and is a five-membered, triaza, diunsaturatedring, said ring optionally mono- or di-substituted with up to twosubstituents independently selected from R⁴ and R⁵;

wherein R¹, R², R³, R⁴ and R⁵ are each independently hydrogen,hydroxy(C₁-C₄)alkyl, (C₁-C₄)alkyl, (C₁-C₄)alkylthio, (C₃-C₄)cycloalkyl,(C₃-C₇)cycloalkyl(C₁-C₄)alkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkoxy(C₁-C₄)alkyl,mono-N- or di-N,N-(C₁-C₄)alkylcarbamoyl, M or M(C₁-C₄)alkyl, any of saidprevious (C₁C₄)alkyl moieties optionally having from one to ninefluorines; said (C₁-C₄)alkyl or (C₃-C₄)cycloalkyl optionally mono-ordi-substituted independently with hydroxy, (C₁-C₄)alkoxy,(C₁-C₄)alkylthio, (C₁-C₄)alkylsulfinyl, (C₁-C₄)alkylsulfonyl,(C₁-C₄)alkyl, mono-N— or di-N,N-(C₁-C₄)alkylcarbamoyl or mono-N— ordi-N,N-(C₁-C₄)alkylaminosulfonyl; and said (C₃-C₄)cycloalkyl optionallyhaving from one to seven fluorines;

wherein M is a partially saturated, fully saturated or fully unsaturatedfive to eight membered ring optionally having one to three heteroatomsselected independently from oxygen, sulfur and nitrogen, or, a bicyclicring consisting of two fused partially saturated, fully saturated orfully unsaturated three to six membered rings, taken independently,optionally having one to four heteroatoms selected independently fromnitrogen, sulfur and oxygen;

said M is optionally substituted, on one ring if the moiety ismonocyclic, or one or both rings if the moiety is bicyclic, on carbon ornitrogen with up to three substituents independently selected from R⁶,R⁷ and R⁸, wherein one of R⁶, R⁷ and R⁸ is optionally a partiallysaturated, fully saturated, or fully unsaturated three to seven memberedring optionally having one to three heteroatoms selected independentlyfrom oxygen, sulfur and nitrogen optionally substituted with(C₁-C₄)alkyl and additionally R⁶, R⁷ and R⁸ are optionally hydroxy,nitro, halo, (C₁-C₄)alkoxy, (C₁-C₄)alkoxycarbonyl, (C₁-C₄)alkyl, formyl,(C₁-C₄)alkanoyl, (C₁-C₄)alkanoyloxy, (C₁-C₄)alkanoylamino,(C₁-C₄)alkoxycarbonylamino, sulfonamido, (C₁-C₄)alkylsulfonamido, amino,mono-N- or di-N,N-(C₁-C₄)alkylamino, carbamoyl, mono-N- ordi-N,N-(C₁-C₄)alkylcarbamoyl, cyano, thiol, (C₁-C₄)alkylthio,(C₁-C₄)alkylsulfinyl, (C₁-C₄)alkylsulfonyl, mono-N- ordi-N,N-(C₁-C₄)alkylaminosulfonyl, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl or(C₅-C₇)cycloalkenyl,

wherein said (C₁-C₄)alkoxy, (C₁-C₄)alkyl, (C₁-C₇)alkanoyl,(C₁-C₄)alkylthio, mono-N- or di-N,N-(C₁-C₄)alkylamino or(C₃-C₇)cycloalkyl R⁶, R⁷ and R⁸ substituents are optionallymono-substituted independently with hydroxy, (C₁-C₄)alkoxycarbonyl,(C₃-C₇)cycloalkyl, (C₁-C₄)alkanoyl, (C₁-C₄)alkanoylamino,(C₁-C₄)alkanoyloxy, (C₁-C₄)alkoxycarbonylamino, sulfonamido,(C₁-C₄)alkylsulfonamido, amino, mono-N- or di-N,N-(C₁-C₄)alkylamino,carbamoyl, mono-N- or di-N,N-(C₁-C₄)alkylcarbamoyl, cyano, thiol, nitro,(C₁-C₄)alkylthio, (C₁-C₄)alkylsulfinyl, (C₁-C₄)alkylsulfonyl or mono-N-or di-N,N-(C₁-C₄)alkylaminosulfonyl or optionally substituted with oneto nine fluorines. (See PCT patent applciation number PCT/IB99/00206)

Each of the NHE-1 inhibitors referenced above and other NHE-1 inhibitorscan be used in combination with the compounds of the present inventionto treat or prevent diabetes, insulin resistance, diabetic neuropathy,diabetic nephropathy, diabetic retinopathy, cataracts, hyperglycemia,hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia,atherosclerosis, or tissue ischemia.

The examples presented below are intended to illustrate particularembodiments of the invention, and are not intended to limit the scope ofthe specification, including the claims, in any manner. All patents,patent applications, and other references cited in this application arehereby incorporated by reference.

EXAMPLES Chemical Examples

Exemplary processes for the manufacture of the compounds of theinvention are provided below and are illustrated by reaction schemes.These processes may be carried out in sequential or convergent syntheticroutes. Purification procedures include crystallization and normal phaseor reverse phase chromatography.

As a general note, the preparation of the compounds described herein mayrequire protection of remote functionality (e.g., primary amine,secondary amine, carboxyl). The need for such protection will varydepending on the nature of the remote functionality and the conditionsof the preparation methods. The need for such protection is readilydetermined by one skilled in the art. The use of suchprotection/deprotection methods is also within the skill in the art. Fora general description of protecting groups and their use, see T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, JohnWiley & Sons, New York, 1991.

The following abbreviations are used herein.

Et ethyl DMF dimethylformamide BOC tert-butyloxycarbonyl CBzbenzyloxycarbonyl Ph phenyl h hours d days min minutes equivequivalent(s) DMSO dimethlysulfoxide dec decomposes mp melting pointCIMS chemical ionization mass spectrometry

The bicyclic pyrrolyl acids of Formula 5 can be made by severalsynthetic methods. With regard to Scheme I, a preferred method(Hemetsberger, H. et al., Monatshefte fur Chemie, 103: 194-204 (1972)),begins with condensation of an azido-acetic acid alkyl ester with analdehyde of Formula 2 in an alcoholic solvent in the presence of analkoxide. The alcohol and alkoxide preferred are those derived from thealkyl ester to avoid transesterification problems. The reaction isperformed at a temperature of about −20° C. to about 25° C. for about1-24 hours, generally employing 3-8 equivalents of the alkoxide and anequimolar quantity of the azido-acetic acid alkyl ester. The resultantazides are then heated at reflux in an inert solvent such as xylenes toafford the heterocyclopyrrole esters of Formula 3. An example of asuitable preparation is shown by Procedure H below.

The aldehydes of Formula 2 Can be made by conventional methods known tothose skilled in the art, or methods for their preparation can readilybe determined from the literature (See, for example, Ortiz, J. A. etal., Eur. J. Med. Chem., 23: 477-482 (1988)). With regard to Scheme II,exemplary preparations include Villsmeyer-Haack formylation ofheterocycles (R″=H) of Formula 1 (See, 0. Meth-Cohn and S. P. Stanforthin Comprehensive Organic Synthesis: Selectivity, Strategy & Efficiencyin Modern Organic Chemistry Vol. 2, Pergamon, New York, 1991, C. H.Heathcock, Ed., p 777), metal-halogen exchange of bromo- oriodoheterocycles (R″=Br,I) of Formula 1 or lithiation of heterocycles ofFormula 1 (R″=H) followed by treatment of aryl lithiums of Formula 11with a formylating agent such as dimethylformamide (Ortiz, J. A. et al.,Eur. J. Med. Chem., 23: 477-482 (1988)) or N-methyl formanilide.(See, D.Comins & S. P. Joseph in Encyclopedia of Reagents for Organic SynthesisVol. 5, Wiley, New York, 1995, L. A. Paquette, Ed., p 3503), reductionof heterocyclic esters of Formula 12 (R=alkyl) or acids (R=H) to Formula13 alcohols or aldehydes of Formula 2 (Nicolaou, K. C. et al., Angew.Chem. Int. Ed. Engl., 36: 166-7 (1997)) with reducing agents(Comprehensive Organic Synthesis: Selectivity, Strategy & Efficiency inModern Organic Chemistry Vol 8, I. Fleming, Ed., Pergamon, 1991, NewYork) such as lithium aluminum hydride, diisobutylaluminum hydride, orborane and subsequent oxidation of alcohols of Formula 13 to aldehydesof Formula 2 using oxidizing agents (Comprehensive Organic Synthesis:Selectivity, Strategy & Efficiency in Modern Organic Chemistry Vol 7, S.V. Ley, Ed., Pergamon, 1991, New York) such as pyridiniumchlorochromate, manganese dioxide, Swern reagent, and barium oxide, orhalogenation of the aldehydes of Formula 14 using electrophilic halidesources such as N-halosuccinimide (R. M. Kellogg et al., J. Org. Chem.,33: 2902-2909 (1968)), N-fluoropyridinium salts (Umemoto, T. et al., J.Am. Chem. Soc., 112: 8563-75 (1990)), or elemental halogen (Ortiz, J. A.et al., Eur. J. Med. Chem., 23: 477-482 1988)).

Alternatively, substitution of the heterocyclopyrroles of Formula 3 Canbe accomplished by analogous conventional methods known to those skilledin the art or substitution methods can readily be determined from theliterature. For example, with regard to Scheme III, mono- and bis-halidesubstitution can be accomplished by treatment with an electrophilichalide source such as the N-halosuccinimide, N-fluoropyridinium salts,or elemental halogen (Gale, W. W. et al., J. Org. Chem., 29: 2160-2165(1964)) to produce heterocyclopyrroles of Formula 4 (R′,R′″=H and/orhalide). Methyl substitution can be accomplished by Villsmeyer-Haackformylation to aldehydes of Formula 4 (R′″=CHO) followed by completereduction of the formyl group under various reducing conditions such assodium cyanoborohydride in the presence of zinc iodide in dichloroethane(C. K. Lau et. al., J. Org. Chem., 51: 3038-3043 (1964)). Methyl andother alkyl substitution can also be accomplished by coupling Formula 3bromo- or iodoheterocyclopyrroles (R=Br, I) with alkyl metals such asalkyl copper reagents (Corey, E. J. et al., J. Am. Chem. Soc. 89:3911-12 (1967)). Alkenes and alkynes, in the presence of copper saltssuch as copper iodide (J. M. Tour et al., J. Org. Chem. 61: 6906-6921(1996); G. M. Whitesides et al., J. Org. Chem., 53: 2489-2496 (1988)),and alkenyl and alkynyl stannanes (Stille, J. K., Angew. Chem. Int. Ed.Engl., 25: 508-524 (1986)) can also be coupled to the bromo- oriodoheterocyclopyrroles of Formula 3 (R=Br, I) in the presence of acatalyst such as palladium. Palladium catalysts include but are notlimited to palladium chloride, dichlorobis(triphenylphosphine)palladium(II), tetrakis(triphenylphosphine)palladium (0), and palladium acetate.Other exemplary conditions useful for forming carbon bonds to aromaticrings are described by K. Tamao, D. W. Knight, and K. Sonogashira inComprehensive Organic Synthesis: Selectivity, Strategy & Efficiency inModern Organic Chemistry Vol 3 (Pergamon, New York, 1991, G. Pattenden,Ed., pp 435-551). Condensation of hydroxylamine with formylated estersof Formula 4 (R′″=CHO) or acids of Formula 5 (R=CHO) can either directly(Ford, R. E. et al., J. Med. Chem., 29, 538-549 (1986)) or after asecond dehydration step (Malicorne, G. et al., Eur. J. Med. Chem. Chim.Ther. 26: 3-11 (1991) afford nitrites. Alternatively, nitrilesubstitution can be accomplished by coupling cuprous cyanide to thebromo- or iodoheterocyclopyrroles of Formula 3 (R=Br, I) indimethylformamide (Klemm, L. H. et al., J. Heterocyclic Chem., 21: 785-9(1984)). Other exemplary conditions useful for forming nitrites aredescribed by R. Grashey in Comprehensive Organic Synthesis: Selectivity,Strategy & Efficiency in Modern Organic Chemistry Vol 6 (Pergamon, NewYork, 1991, E. Winterfeldt, Ed., p 225). An example of a suitablenitrile preparation is Procedure G below.

Alternatively the aformentioned methods of substituting theheterocyclopyrroles of Formula 3 Can also be applied to the amides ofFormulas 6 and 7.

The coupling of an acid of Formula 5 (Scheme I) with an amine of FormulaA (Scheme IV) or P (Scheme VII) to make a compound of the presentinvention can be accomplished in several ways, which are analogous tothose well known to those skilled in the art.

In a typical coupling procedure, the acid and amine are combined with asuitable coupling agent. A suitable coupling agent is an agent thattransforms the carboxylic acid group into a reactive species such thatan amide linkage is formed between the carboxylic acid and the amine.

The coupling agent can provide for the coupling in a one pot process orseveral steps may be required to achieve the coupling. Examples ofsuitable coupling agents include1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride-hydroxybenzotriazole (DEC/HBT), carbonyldiimidazole,dicyclohexylcarbodiimide/hydroxybenzotriazole,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ),carbonyldiimidazole/HBT, propanephosphonic anhydride (propanephosphonicacid anhydride, PAA) and diethylphosphorylcyanide.

The coupling reaction is generally performed in an inert solvent,preferably an aprotic solvent at a temperature of about −20° C. to about50° C. for about 1 to about 48 hours, optionally in the presence of atertiary amine such as triethylamine. Suitable solvents includeacetonitrile, dichloromethane, ethyl acetate, dimethylformamide andchloroform, or mixtures thereof.

In an example of a multistep coupling process, the carboxylic acid groupis reacted with the coupling agent to form an activated intermediate,which can be isolated in the first step of the process. In a secondstep, the activated intermediate is then reacted with the amine to formthe amide. Examples of coupling agents that convert an acid to anactivated intermediate include thionyl chloride, oxalyl chloride, whichform acid chlorides, cyanuric fluoride, which forms acid flourides, oran alkyl chloroformate such as isobutyl or isopropenyl chloroformate(with a tertiary amine base), which forms a mixed anhydride of thecarboxylic acid. If the coupling agent is oxalyl chloride, it isadvantageous to employ a small amount of dimethylformamide as acosolvent with another solvent such as dichloromethane to catalyze theformation of the acid chloride. The acid chloride may be coupled withthe amine in an appropriate solvent and a suitable base. Acceptablesolvent/base combinations include dichloromethane, dimethylforamide oracetonitirile, or mixture thereof in the presence of a tertiary aminebase such as triethylamine. Other appropriate solvent/base combinationsinclude water or a C₁-C₅ alcohol, or mixtures thereof, together with acosolvent such as dichloromethane, tetrahydrofuran or dioxane, and abase such as sodium or potassium carbonate, sodium, potassium or lithiumhydroxide, or sodium bicarbonate in sufficient quantity to consume theacid liberated in the reaction. Use of a phase transfer catalyst(typically 1 to 10 mole %) such as a quaternary ammonium halide (e.g.,tetrabutylammonium bromide or methyl trioctylammonium chloride) isadvantageous when a mixture of only partially miscible cosolvents isemployed (e.g. dichloromethane-water or dichloromethane-methanol). Useof these coupling agents and appropriate selection of solvents andtemperatures are known to those skilled in the art and can be readilydetermined from the literature. These and other exemplary conditionsuseful for coupling carboxylic acids with amines are described inHouben-Weyl, Vol. XV, part 11, E. Wunsch, Ed., G. Thieme Verlag, 1974,Stuttgart, and M. Bodansky, Principles of Peptide Synthesis,Springer-Verlag Berlin 1984, and The Peptides: Analysis, Synthesis andBiology (ed. E. Gross and J. Meienhofer), Vols 1-5 (Academic Press, NY1979-1983).

The amines that are reacted with the carboxylic acid function group tomake an amide of the present invention can be synthesized in a number ofways. With regard to Scheme IV, an alpha amino acid of Formula A can beprotected on the amine nitrogen with an appropriate protecting group(Pr) to form a protected amino acid of Formula B. One skilled in the artcan readily select an appropriate amine protecting group. For example,two common protecting groups are BOC, which is introduced by treatingthe amino acid with di-tert-butyldicarbonate, preferably in a proticsolvent or a solvent mixture at high pH, and CBZ, which is introduced bytreating the amino acid with benzylchloroformate, preferably in a proticsolvent or a solvent mixture, and a base. The amine protected amino acidcompound of Formula B is then coupled with an appropriate amine of theformula HNRR (where the R groups are consistent with the compounds ofthe present invention) in a procedure analogous to the coupling reactionset forth above to form a protected amide compound of Formula C. Theprotected amide of Formula C can then be deprotected to form an amide ofFormula D. If the protecting group is BOC, the deprotection is typicallydone by treating the protected compound with an acid in an aproticsolvent. Suitable acids include HCl, CH₃SO₃H and trifluoroacetic acid.

It may also be desired to make esters of the compounds of Formula A orB. With regard to Scheme V, the esters of compound A and B can be madeby reacting the compound with an appropriate alcohol and an acidcatalyst such as concentrated sulfuric acid or by treatment with analkyl halide such as methyl idodide and a base such as potassiumcarbonate. Compounds of Formula E can also be made by protecting acompound of Formula A, and then forming the ester. Alternatively,compounds of Formula E can be made starting with a compound of FormulaA, forming an ester, and then protecting the amine group. Analogousprocedures for the formation and cleavage of esters and the protectionof amine groups are well known to those skilled in the art.

According to reaction Scheme VI, the compounds of Formula A when R^(b)is not hydrogen can be prepared as follows. The Formula B amino acid canbe prepared by N-alkylation of a compound of Formula G, which is anamine protected alpha amino acid. N-alkylation is well known in the artand can be accomplished using an appropriate alkylating agent and asuitable base. Specific procedures for alkylation are described inBenoiton, Can. J. Chem., 55: 906-910 (1985), and Hansen, J. Org. Chem.,50: 945-950 (1977). For example, when R^(b) is methyl, and Pr is BOC,sodium hydride and methyl iodide in tetrahydrofuran can be used.Deprotection of the compound of Formula B results in a compound ofFormula A.

Alternatively, a compound of Formula H can be N-alkylated by a threestep sequence involving reductive benzylation, such as with benzaldehydefollowed by Pd/C-catalyzed hydrogenation to give the mono-N-benzylderivative, and reductive amination with an appropriate carbonylcompound, for example formaldehyde and sodium cyanoborohydride tointroduce R^(b) as methyl, to give the N-benzyl, substituted amino acid.The N-benzyl protecting group is conveniently removed, for example, byhydrogenation with an appropriate catalyst, to yield a compound ofFormula A. Specific conditions for the three step alkylation procedureare described by Reinhold et al., J. Med. Chem., 11: 258-260 (1968).

While many of the alpha amino acid starting materials are known, theycan be synthesized by a number of procedures that are well known in theart. For example, the Strecker synthesis or variations thereof can beused. Accordingly, an aldehyde, sodium or potassium cyanide and ammoniumchloride react to form an aminonitrile. It is noted that the aldehydeselected is determined by the desired amino acid. The aminonitirle isthen hydrolyzed with a mineral acid to form the desired amino acid.Alternatively, the Bucherer-Berg method may be used where a hydantoin isformed by heating an aldehyde with ammonium carbonate and potassiumcyanide followed by hydrolysis, for example, with barium hydroxide inrefluxing dioxane, with acid or base to form the desired compounds.

Suitable methods for the synthesis and/or resolution of compounds ofFormula H (Scheme VI) (alpha amino acids) are found in reviews byDuthaler, Tetrahedron, 50: 1539-1650 (1994), or by Williams, Synthesisof Optically Active Amino Acids, Pergamon, Oxford, U.K. 1989. Anothermethod is shown in Corey and Link, J. Am. Chem. Soc., 114: 1906-1908(1992).

The synthesis of the compounds of the present invention where Y is

is accomplished by the coupling of an amide compound of Formula P(Scheme VII) with a bicylic pyrrolyl carboxylic acid of Formula 5. Theprocedure for the coupling can be carried out as described above. Thesynthesis of the amides of Formula P is illustrated by Scheme VII. Tobegin, a nitrogen protected amino aldehyde of Formula J is treated withpotassium or sodium cyanide in aqueous solution with a cosolvent such asdioxane or ethyl acetate at a temperature of about 0° C. to about 50° C.to provide a compound of Formula K, which is cyanohydrin. Thecyanohydrin of Formula K is then reacted with an alcohol such asmethanol and a strong acid catalyst such as HCl at a temperature ofabout 0° C. to about 50° C., followed by the addition of water, ifnecessary. The protecting group is then removed, if still present, by anappropriate deprotection method yielding a compound of Formula L. Forexample, if the protecting group is BOC, the Formula L compound isdirectly formed from the Formula K compound, and addition of water isnot necessary. The Formula L compound can be protected on the nitrogento form a compound of Formula M followed by hydrolysis of the ester withaqueous alkali at a temperature of about 0° C. to about 50° C. in areaction-inert solvent resulting in the corresponding hydroxy acid ofFormula N. The hydroxy acid of formula N is coupled to a suitable amineto form the protected amino amide of Formula O, which is thendeptrotected to form a compound of Formula P. An analogous example ofthe conversion of a Formula K compound to the corresponding Formula Lcompound is provided in PCT publication WO/9325574, Example 1a. Otheranalogous examples where a cyanohydrin is converted to a Formula Mcompound can be found in U.S. Pat. No. 4,814,342 and EPO publication0438233.

It may be desirable to have a certain stereochemistry at the alpha andbeta positions of the compounds of Formula P. (The alpha position is thecarbon atom containing the hydroxyl group.) The desired stereochemistrycan be obtained by the use of a single stereoisomeric aldehyde ofFormula J. The Formula K cyanohydrin can be prepared from thestereochemically pure aldehyde by treatment with sodium or potassiumcyanide as described above while maintaining the stereochemistry of thechiral carbon of the aldehyde, resulting in a mixture of stereoisomers,which can be separated, as is well known to those skilled in the art bycrystallization. See, for example, Biochemisrty, 31: 8125-8141 (1992).Alternatively, isomer separation can be effected by chromatography orrecrystallization techniques after conversion of a compound of Formula Kto a compound of Formula L, M, N, O, or P by the procedures describedherein and analogous to those well known in the art.

With reference to Scheme VIII, the aminoaldehydes of Formula J can bemade from the corresponding alpha amino acid of Formula Q. In onemethod, the alpha amino acid of Formula Q is protected on nitrogen andesterified to form a compound of Formula R. The compound of Formula R isreduced, for example, with diisobutylaluminum hydride in hexane ortoluene, or a mixture thereof, at a temperature of about −78° C. toabout −50° C. followed by quenching with methanol at −78° C. asdescribed in J. Med. Chem., 28: 1779-1790 (1985) to form the Formula Jaldehyde.

Alternatively, the Formula J aldehydes can be made by oxidation ofFormula T alcohols, for example, with pyridine-SO₃ at a temperature ofabout −10° C. to about 40° C. in a reaction-inert solvent, preferablydimethylsulfoxide. The protected amino alcohols of Formula T, if notcommercially available, can be made by the protection of aminoalcoholsof Formula S. The Formula S aminoalcohols are prepared by the reductionof amino acids of formula Q. The reduction can be accomplished bytreating Formula Q amino acids with lithium aluminum hydride accordingto the procedure described by Dickman et al., Organic Synthesis; Wiley:New York, 1990; Collect. Vol. VIII, p. 530. or with sulfuric acid-sodiumborohydride by the procedure of Abiko and Masamune, Tetrahedron Lett.,333: 5517-5518 (1992) or with sodium borohydride-iodine according to theprocedure of McKennon and Myers, J.Org. Chem., 58: 3568-3571 (1993),where other suitable procedures are also reviewed. The preparation ofthe alpha amino acid and N-alkylated alpha amino acids has beendescribed above.

In addition, PCT publications WO 96/3985, published Dec. 12, 1996, andWO 96/39384, published Dec. 12, 1996, contain further details andexemplifications of the processes of synthesizing aspects of the presentcompounds. These publications are hereby incorporated by reference.

Equipment and General Procedures

NMR spectra were recorded on a Bruker AM300 or Varian XL-400spectrometer at about 23° C. at 300 or 400 MHz, respectively, for protonnuclei. Unless otherwise specified, NMR spectral data is reported for a400 MHz spectrometer. Routine mass spectral data were obtained using aVG/Fisons Instruments Platform II spectrometer operating with anAtmospheric Pressure Chemical Ionization (APCI) source. Melting pointsare uncorrected and were determined on a Thomas Hoover capillary meltingpoint apparatus. Unless otherwise specified, reagents were used asobtained from commercial sources. The term “concentrated” refers toremoval of solvent on a rotary evaporator. Exceptions in the use of theProcedures A-H are noted individually in parentheses, following mentionof the procedure.

General Synthetic Procedures

Procedure A (Amide Formation Using 1-Hydroxybenzotriazole Hydrate and1-(3-Dimethylamino-propyl)-3-ethylcarbodiimide Hydrochloride)

A 0° C. 0.1-0.7 M mixture the primary amine (1 equiv, or a primary aminesalt and 1 equiv of triethylamine per equiv HCl), 1 equiv of thespecified carboxylic acid, and 1 equiv of 1-hydroxybenzotriazole hydrate(1 equiv relative to the carboxylic acid), in 3:1dichloromethane:dimethylformamide is treated with 1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride. Themixture is allowed to warm to room temperature over several hours,stirred overnight, concentrated to remove the dichloromethane, andpartitioned between ethyl acetate and 1-2 N HCl. The aqueous phase isextracted with ethyl acetate. The combined organic phases are washedwith saturated aqueous NaHCO₃, dried over MgSO₄, and concentrated givingcrude product which is purified by chromatography on silica gel and/orrecrystallization.

Procedure B (Amide Formation Using 1-Hydroxy-7-azabenzotriazole Hydrateand 1-(3-Dimethylamino-propyl)-3-ethylcarbodiimide Hydrochloride)

A 0° C. 0.1-0.3 M mixture of the primary amine or primary amine salt (1equiv), 1 equiv of triethylamine, 1 equiv of the specified carboxylicacid, and 1 equiv of 1-hydroxy-7-azabenzotriazole (1 equiv relative tothe carboxylic acid), in dimethylformamide is treated with 1 equiv(corresponding in mol ratio to the carboxylic acid)1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride. Themixture is allowed to warm to room temperature over several hours,stirred overnight, and partitioned between ethyl acetate and 1-2 N HCl.The organic phase is washed with saturated aqueous NaHCO₃, dried overMgSO₄, and concentrated giving crude product which is purified bychromatography on silica gel.

Procedure C (Amide Formation Using 1-Hydroxy-7-azabenzotriazole Hydrateand 1-(3-Dimethylamino-propyl)-3-ethylcarbodiimide Methiodide)

A 0.3 M mixture of the primary amine hydrochloride (1 equiv), 1.2 equivof triethylamine, 1 equiv of the specified carboxylic acid, and 1.2equiv of 1-hydroxybenzotriazole hydrate in dimethylformamide is treatedwith 1.2 equiv 1-(3-dimethylamino-propyl)-3-ethylcarbodiimidemethiodide. The mixture is stirred overnight and partitioned betweenethyl acetate and 1 N NaOH. The organic phase is washed sequentiallywith 1 N HCl and water, dried over MgSO₄, and concentrated giving crudeproduct.

Procedure D (Hydrolysis of Ethyl Ester with Potassium Hydroxide)

A 0.1-0.8 M suspension of the ethyl ester (1 equiv) and KOH (2 equiv) inwater is heated at reflux for 1-7 h, allowed to cool to roomtemperature, stirred overnight, and extracted with ethyl acetate. Theaqueous phase is acidified with 2 N HCl and extracted with ethylacetate. The combined organic phases are dried over MgSO₄, andconcentrated giving crude product which is purified by chromatographyand/or washing with solvent.

Procedure E (Hydrolysis of Ethyl Ester with Sodium Hydroxide)

A 0.1-0.8 M suspension of the ethyl ester (1 equiv) and 2 N NaOH (10equiv) in methanol is heated at 65° C. for 2 h, allowed to cool to roomtemperature, concentrated to remove the methanol, diluted with water,and extracted with ethyl acetate. The aqueous phase is acidified with 2N HCl and extracted with ethyl acetate. The combined organic phases aredried over MgSO₄, and concentrated giving crude product which ispurified by recrystallization.

Procedure F (Hydrolysis of Ethyl Ester with Lithium Hydroxide)

A 0.1-0.3 M solution of the ethyl ester (1 equiv) and LiOH—H₂O (4-6equiv) in 3:2:1 tetrahydrofuran:methanol:water is heated at 60-65° C.overnight, allowed to cool to room temperature, concentrated to removethe tetrahydrofuran and methanol, and acidified with 1-2 N HCl. Theresultant precipitate is filtered, washed with water, and dried in vacuogiving product.

Procedure G (Nitrile Formation with Hydroxylamine Hydrochloride)

A 0.1-0.2 M mixture of the aldehyde (1 equiv) and hydroxylaminehydrochloride (2.2-4 equiv) in dimethylformamide is heated at 125° C.overnight, allowed to cool to room temperature, and partitioned betweenethyl acetate and water. The aqueous phase is extracted with ethylacetate. The combined organic phases are washed with water, dried overMgSO₄, and concentrated giving crude product which is purified bychromatography on silica gel.

Procedure H (Annulation with Azido-acetic Acid Ethyl Ester)

A 0° C. 0.6-1.2 M solution of sodium (3-4 equiv) in ethanol is treatedwith a mixture of the aldehyde (1 equiv) and azido-acetic acid ethylester (1 equiv relative to sodium) dropwise such that the reactiontemperature was maintained at 5-10° C. The reaction mixture is stirredfor 1-2 h, quenched with cold saturated aqueous NH₄Cl, and extractedwith ether. The combined organic phases are dried over MgSO₄ andconcentrated. The residue is purified by chromatography on silica gel. A0.1-0.2 M solution of the resultant acrylate in xylenes is heated atreflux for 20-60 min and allowed to cool to room temperature. Thereaction solution is either cooled further to induce crystallization ofthe product or concentrated giving crude product which is purified bywashing with hexanes and/or chromatography on silica gel.

Example 1

6H-Thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

6H-Thieno[2,3-b]pyrrole-5-carboxylic acid (Soth, S. et al., Bull. Soc.Chim. Fr., 2511-2515 (1975)) and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure A (4-(dimethylamino)pyridine (0.1equiv) also added to the reaction mixture).

mp 137-145° C.; CIMS m/e 430.2 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.67 (br s, 1H), 7.74 (d, J=8.9 Hz, 1H), 7.21 (m,4H), 7.11 (m, 1H), 6.96 (s, 3H), 5.03 (dd, J=2.9, 7.5 Hz, 0.5H), 4.93(m, 1H), 4.87 (m, 0.5H), 4.80 (dd, J=2.9, 7.5 Hz, 0.5H), 4.74 (br s,0.5H), 4.40 (br s, 1H), 4.19 (m, 1H), 4.06 (dq, J=3.2, 5.3 Hz, 0.5H),3.99-3.87 (m, 1.5H), 3.54 (m, 1H), 3.38 (m, 0.5H), 3.25-3.06 (m, 2.5H),2.94-2.81 (m, 2H).

Example 1a

[(1S)-Benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-carbamicacid benzyl ester

(2R,3S)-3-Benzyloxycarbonylamino-2-hydroxy-4-phenyl-butyric acid(Takita, T. et al., J. Med. Chem., 20: 510-515 (1977)) andpyrrolidine-(3R,4S)-diol hydrochloride were coupled according toProcedure A (dimethylformamide reaction solvent concentrated to ½ volumebefore work-up).

CIMS m/e 415.2 (MH⁺).

¹H NMR (DMSO-d₆) δ 7.28-7.15 (m, 10H), 7.07-7.01 (m, 1H), 4.94-4.75 (m,4.5H), 4.65 (d, J=7.7 Hz, 0.5H), 4.09-3.88 (m, 4H), 3.51-3.38 (m, 1H),3.27-3.07 (m, 3H), 2.83-2.63 (m, 2H).

Example 1b

(3S)-Amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-one

According to a procedure by Takita, T. et al. (J. Med. Chem., 20:510-515 (1977)) a mixture of[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-carbamicacid benzyl ester (1.2 g, 2.9 mmol) and 10% palladium on carbon (120 mg)in methanol (20 mL) was shaken under a hydrogen atmosphere (40-45 psi)on a Parr apparatus overnight, filtered through Celite®, andconcentrated. The product was obtained as a sticky solid (1.0 g, 100%).

CIMS m/e 281.2 (MH⁺).

¹H NMR (DMSO-d₆) δ 7.27-7.13 (m, 5H), 4.95-4.80 (m, 3H), 3.93 (br s,2H), 3.83 (dd, J=3.3, 9.1 Hz, 1H), 3.45-3.05 (m, 6H), 2.99 (dq, J=3.5,6.3 Hz, 1H), 2.65 (m, 1H), 2.50 (m, 1H).

Example 2

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure A.

mp 143-145° C.; CIMS m/e 508.0/510.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.72 (brs, 1H), 7.84 (d, J=9.1 Hz, 1H), 7.22 (m,5H), 7.11 (m, 1H), 6.99 (s, 1H), 5.04 (d, J=7.3 Hz, 0.5H), 4.95 (m, 1H),4.89 (d, J=5.0 Hz, 0.5H), 4.80 (d, J=7.7 Hz, 0.5H), 4.75 (d, J=4.4 Hz,0.5H), 4.40 (m, 1H), 4.19 (m, 1H), 4.00-3.85 (m, 2H), 3.54 (m, 1H), 3.39(dd, J=4.9, 12.6 Hz, 0.5H), 3.22 (m, 1.5H), 3.16-3.06 (m, 1H), 2.94-2.81(m, 2H).

Example 2a

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (Eras, J.;Galvez, C.; Garcia, F., J. Heterocycl. Chem., 21: 215-217 (1984)) washydrolyzed according to Procedure F.

CIMS m/e 244.0/246.0 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.66 (brs, 1H), 12.10 (brs, 1H), 7.22 (s, 1H), 6.87(d, J=2.1 Hz, 1H).

Example 3

2-Methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-Methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethyl/carbodiimide hydrochloride, 15:1dichloromethane:dimethylformamide; combined organic phases washed withwater prior to saturated aqueous NaHCO₃).

mp 154-157° C.; CIMS m/e 442.2 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 11.58 (m, 1H), 7.66 (d, J=8.5 Hz, 1H), 7.22-7.10 (m,5H), 6.86 (s, 1H), 6.64 (s, 1H), 5.03-4.73 (m, 3H), 4.38 (br s, 1H),4.18 (m, 1H), 3.98-3.88 (m, 2H), 3.53 (m, 1H), 3.39-3.05 (m, 3H),2.90-2.83 (m, 2H), 2.38 (s, 3H).

Example 3a

2-Methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester

Using a procedure by C. K. Lau et. al. (J. Org. Chem., 51: 3038-3043(1986)), a mixture of 2-formyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acidethyl ester (Soth, S. et al., Bull. Soc. Chim. Fr., 2511-2515 (1975);500 mg, 2.24 mmol), ZnI₂ (1.08 g, 3.36 mmol), and NaBH₃CN (1.06 g, 16.8mmol) in dichloroethane (25 mL) was stirred for 7 d and quenched withsaturated aqueous NH₄Cl (25 mL). The resultant biphasic mixture wasstirred for an additional 30 min, extracted with ethyl acetate, driedover Na₂SO₄, and concentrated. The product was purified byChromatotron-chromatography (3:2 hexanes:ether) and obtained as a whitefoam (233 mg, 50%).

mp 107-109° C.; CIMS m/e 208.3 (MH⁺).

¹H NMR (CDCl₃) δ 9.13 (br s, 1H), 6.94 (s, 1H), 6.61 (s, 1H), 4.33 (q,J=7.1 Hz, 2H), 2.48 (s, 3H), 1.36 (t, J=7.1 Hz, 3H).

Example 3b

2-Methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid

2-Methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure E.

mp 180-182° C. dec.; CIMS m/e 180.1 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.36 (s, 1H), 11.93 (s, 1H), 6.77 (s, 1H), 6.66 (s,1H), 2.40 (s, 3H), 1.36 (t, J=7.1 Hz, 3H).

Example 3c

[(1S)-Benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-carbamicacid Tert-Butyl Ester

(2R,3S)-3-tert-Butoxycarbonylamino-2-hydroxy-4-phenyl-butyric acid andpyrrolidine-(3R,4S)-diol hydrochloride were coupled according toProcedure A (1.05 equiv triethylamine, 1.1 equiv carboxylic acid; 1.5equiv 1-hydroxybenzotriazole hydrate; 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride; afterdichloromethane removal, residue partitioned between ethyl acetate and 2N NaOH; combined organic phases washed sequentially with 2 N HCl andsaturated NaCl).

CIMS m/e 381 (MH⁺).

Example 3d

(3S)-Amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-oneHydrochloride

To a 0° C. solution of[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-carbamicacid tert-butyl ester (1.1 g, 2.9 mmol) in methanol (4 mL) was added 4 NHCl in dioxane (7.2 mL, 28.9 mmol). The solution was allowed to slowlywarm to room temperature and stirred overnight. The reaction mixture wasconcentrated and the residue was washed with methanol and dried invacuo. The product was obtained as a white solid (1.03 g, 113%).

CIMS m/e 281.2 (MH⁺).

Example 4

(±)-2-Methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[1-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Methyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(±)-2-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 15:1dichloromethane:dimethylformamide; combined organic phases washed withwater prior to saturated aqueous NaHCO₃).

mp 134-136° C.; CIMS m/e 412.0 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 11.60 (s, 1H), 8.37 (m, 1H), 7.27-6.98 (m, 6H), 6.65(s, 1H), 4.99-4.73 (m, 3H), 4.05-3.82 (m, 2.5H), 3.40-2.87 (m, 5.5H),2.38 (s, 3H).

Example 4a

(±)-[1-Benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-Carbamicacid Tert-Butyl Ester

Boc-DL-Phenylalanine and pyrrolidine-(3R,4S)-diol hydrochloride werecoupled according to Procedure A (1.5 equiv 1-hydroxybenzotriazolehydrate, 1.1 equiv 1-(3-dimethylamino-propyl)-3-ethylcarbodiimidehydrochloride, dichloromethane; 3 d reaction time).

CIMS m/e 351.2 (MH⁺).

¹H NMR (CDCl₃) δ 7.28-7.19 (m, 5H), 5.35 (m, 1H), 4.52 (m, 1H),4.14-3.99 (m, 1.5H), 3.78-3.63 (m, 1.5H), 3.46-3.34 (m, 2H), 3.00-2.65(m, 3H), 1.40 (s, 9H).

Example 4b

(±)-2-Amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-oneHydrochloride

To a 0° C. solution of(±)-[1-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-carbamicacid tert-butyl ester (6.5 g, 20 mmol) in methanol (8 mL) was added 4 NHCl in dioxane (50 mL, 200 mmol). The solution was allowed to slowlywarm to room temperature and stirred overnight. The resultant whitereaction mixture was diluted with ether and the precipitate wasfiltered, washed with ether, and dried in vacuo. The product wasobtained as a white solid (5 g, 87%).

CIMS m/e 251.2 (MH⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 8.28 (br s, 3H), 7.38-7.21 (m, 5H),5.11-4.93 (m, 2H), 4.34-4.22 (m, 1H), 3.96 (m, 1H), 3.81-3.70 (m, 1H),3.89 (m, 0.5H), 3.47 (m, 0.5H), 3.33-2.85 (m, 4H), 2.63 (m, 1H).

Example 5

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-11-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide; combined organic phases washed withwater prior to saturated aqueous NaHCO₃).

mp 140-142° C.; CIMS m/e 477.9/479.9 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.73 (s, 1H), 8.55 (d, J=8.1 Hz, 1H), 7.26-7.09 (m,7H), 5.00 (br s, 0.5H), 4.91-4.85 (m, 1.5H), 4.77 (m, 1H), 4.07-3.93 (m,1.5H), 3.83 (m, 1.5H), 3.41-3.25 (m, 1H), 3.13 (m, 2H), 3.00-2.87 (m,2H).

Example 5a

[(1S)-Benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-vi)-2-oxo-ethyl]-carbamicacid tert-butyl Ester

Boc-L-Phenylalanine and pyrrolidine-(3R,4S)-diol hydrochloride werecoupled according to Procedure A (1.5 equiv 1-hydroxybenzotriazolehydrate, dichloromethane; reaction mixture diluted with ethyl acetateand washed sequentially with 1 N NaOH, 1 N HCl, and saturated sodiumchloride prior to drying).

CIMS m/e 351.2 (MH⁺).

¹H NMR (DMSO-d₆) δ 7.25-7.13 (m, 5H), 7.06 (dd, J=8.4, 13.6 Hz, 1H),4.98 (d, J=5.4 Hz, 0.5H), 4.91 (d, J=5.0 Hz, 0.5H), 4.84 (m, 1H), 4.25(dd, J=8.5, 14.3 Hz, 1H), 4.02 (m, 0.5H), 3.94 (m, 0.5H), 3.79 (m, 1H),3.68 (dd, J=5.9, 10.1 Hz, 0.5H), 3.38 (dd, J=5.3, 12.2 Hz, 0.5H),3.27-3.10 (m, 3H), 2.83-2.67 (m, 2H), 1.27 (s, 5H), 1.25 (s, 4H).

Example 5b

(2S)-Amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-oneHydrochloride

To 4 N HCl in dioxane (120 mL, 480 mmol) was added[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-carbamicacid tert-butyl ester (27 g, 77 mmol). The solution was stirred 2.5 hand concentrated. The product was obtained as a white solid (21.5 g,98%).

CIMS m/e 251.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 8.33 (br s, 3H), 7.32-7.16 (m, 5H), 5.10-4.86 (m,2H), 4.25-4.13 (m, 1H), 3.93 (m, 1H), 3.73-3.66 (m, 1H), 3.54 (m, 0.5H),3.46-3.23 (m, 1.5H), 3.18-3.05 (m, 2H), 3.00 (m, 0.5H), 2.91-2.79 (m,1H), 2.57 (dd, J=5.6, 10.0 Hz, 0.5H).

Example 6

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide; combined organic phases washed withwater prior to saturated aqueous NaHCO₃).

mp 148-152° C.; CIMS m/e 464.0/465.9 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.71 (m, 1H), 7.84 (d, J=8.9 Hz, 1H), 7.23-6.98 (m,7H), 5.05-4.74 (m, 3H), 4.39 (m, 1H), 4.20 (m, 1H), 4.02-3.88 (m, 2H),3.54 (m, 0.5H), 3.41-3.06 (m, 3.5H), 2.94-2.83 (m, 2H).

Example 6a

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester

Using a modified procedure by R. M. Kellogg et al. (J. Org. Chem., 33:2902-290 (1968)), to a 0° C. solution of6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (Eras, J.; Galvez,C.; Garcia, F., J. Heterocycl. Chem., 21: 215-217 (1984); 1.45 g, 7.44mmol) in acetic acid (15 mL) and CHCl₃ (15 mL) was addedN-chlorosuccinimide (1.04 g, 7.81 mmol) over 2 h. The reaction mixturewas slowly allowed to warm to room temperature over several hours,stirred overnight, concentrated to remove the chloroform, diluted withwater, basified with 5 N NaOH, and extracted with ethyl acetate. Thecombined organic phases were washed with saturated aqueous NaHCO₃, driedover MgSO₄, and concentrated. The product was purified by chromatronchromatography (radial) using 90:10 hexanes/diethyl ether and then thenthen the product obtained was recrystallized using hexanes/diethyl ether(90:10). Last, the product of the recrystallization was further purifiedby flash column chromatography using 90:10 petroleum ether/isopropylether. The resulting product was obtained as a white solid (824 mg,48%).

CIMS m/e 228.2/230.2 ((M−H)⁺).

¹H NMR (CDCl₃) δ 9.28 (br s, 1H), 6.98 (d, J=1.9 Hz, 1H), 6.88 (s, 1H),4.33 (q, J=7.2 Hz, 2H), 1.35 (t, J=7.2 Hz, 3H).

Example 6b

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure D (reaction heated at 85° C.).

CIMS m/e 200.1/202.1 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.66 (br s, 1H), 12.08 (s, 1H), 7.11 (d, J=1.9 Hz,1H), 6.86 (t, J=2.1 Hz, 1H).

Example 7

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide; combined organic phases washed withwater prior to saturated aqueous NaHCO₃).

mp 142-145° C.; CIMS m/e 432.1/434.2 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 11.72 (m, 1H), 8.55 (d, J=8.5 Hz, 1H), 7.28-7.10 (m,7H), 5.00 (d, J=5.2 Hz, 0.5H), 4.99-4.70 (m, 2.5H), 4.09-3.76 (m, 2.5H),3.41-3.24 (m, 2H), 3.13 (m, 1.5H), 3.02-2.87 (m, 2H).

Example 8

2,4-Dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2,4-Dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide, 0.06 M).

mp 130-134° C. (dec.); CIMS m/e 496.2/498.2 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.13 (br s, 1H), 7.40-7.15 (m, 7H), 5.51 (d, J=5.8Hz, 0.5H), 5.38 (d, J=6.3 Hz, 0.5H), 4.99 (d, J=4.9 Hz, 1H), 4.92 (d,J=4.8 Hz, 0.5H), 4.85 (d, J=4.1 Hz, 0.5H), 4.55-4.40 (m, 1H), 4.26 (m,1H), 4.08-3.90 (m, 2H), 3.56-2.89 (m, 6H).

Example 8a

2,4-Dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester

To a 0° C. solution of 6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethylester (180 mg, 0.92 mmol) in acetic acid (2 mL) and CHCl₃ (2 mL) wasadded N-chlorosuccinimide (294 mg, 2.2 mmol) over 30 min. The reactionmixture was slowly allowed to warm to room temperature over severalhours, stirred overnight, concentrated to remove the chloroform, dilutedwith water, basified with 5 N NaOH, and extracted with ethyl acetate.The combined organic phases were washed with saturated aqueous NaHCO₃,dried over MgSO₄, and concentrated. The product was purified byChromatron-chromatography (4:1 hexanes:ether) and obtained as a whitesolid (180 mg, 74%).

mp 166-167° C.; CIMS m/e 262.1/264.1 ((M−H)⁺).

¹H NMR (300 MHz, CDCl₃) δ 9.21 (br s, 1H), 6.90 (s, 1H), 4.37 (q, J=7.2Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

Example 8b

2,4-Dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid

2,4-Dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure E (reflux for 12 h before allowing tocool to room temperature; acidification with concentrated HCl; nopurification).

CIMS m/e 234.0/236.0 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.28 (br s, 1H), 7.17 (s, 1H).

Example 9

(±)-4H-Thieno[3,2-b]pyrrole-5-carboxylic acid[1-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

4H-Thieno[3,2-b]pyrrole-5-carboxylic acid (Soth, S.; Farnier, M.;Paulmier, C., Can. J. Chem. 56, 1429-34 (1978)) and(+)-2-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (9:1dichloromethane:dimethylformamide, 0.06 M; combined organic phaseswashed with 2 N NaOH, dried over Na₂SO₄).

mp 212° C.; CIMS m/e 400.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.59 (m, 1H), 8.51 (d, J=8.5 Hz, 1H), 7.36 (d, J=5.2Hz, 1H), 7.30-7.11 (m, 5H), 6.92 (m, 1H), 5.01 (d, J=5.0 Hz, 0.5H), 4.92(d, J=4.8 Hz, 0.5H), 4.87-4.76 (m, 2H), 4.04-3.93 (m, 1H), 3.83 (m,1.5H), 3.43-3.25 (m, 2.5), 3.13 (m, 1H), 3.03-2.86 (m, 2H).

Example 10

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onehydrochloride were coupled according to Procedure B (1.5 equiv1-hydroxy-7-azabenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide; reaction mixture stirred for 5 d,concentrated to remove dichloromethane before work-up; combined organicphases washed with water prior to saturated aqueous NaHCO₃).

mp 138-143° C.; CIMS m/e 508.0/510.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.68 (m, 1H), 7.86 (d, J=9.1 Hz, 1H), 7.25-7.18 (m,4H), 7.12-7.08 (m, 2H), 7.02 (s, 1H), 5.05 (d, J=7.5 Hz, 0.5H), 4.95 (m,1H), 4.88 (d, J=5.0 Hz, 0.5H), 4.81 (d, J=7.5 Hz, 0.5H), 4.75 (d, J=3.5Hz, 0.5H), 4.46-4.37 (m, 1H), 4.20 (m, 1H), 4.08-3.85 (m, 2H), 3.54 (m,1H), 3.40-3.05 (m, 3H), 2.95-2.81 (m, 2H).

Example 10a

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (Eras, J.;Galvez, C.; Garcia, F., J. Heterocycl. Chem., 21: 215-217 (1984)) washydrolyzed according to Procedure D (after cooling to room temperature,acidification with 2 N HCl; resultant precipitate filtered, suspended intoluene, concentrated; no purification).

CIMS m/e 244.0/246.0 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.63 (s, 1H), 12.04 (s, 1H), 7.13 (s, 1H), 6.97 (s,1H).

Example 11

4H-Thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

4H-Thieno[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onehydrochloride were coupled according to Procedure A (combined organicphases washed with 2 N NaOH, dried over Na₂SO₄).

mp 185-190° C.; CIMS m/e 430.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.57 (s, 0.5H), 11.53 (s, 0.5H), 7.80 (d, J=8.9 Hz,1H), 7.32 (dd, J=0.9, 5.3 Hz, 1H), 7.23 (m, 4H), 7.12 (m, 1H), 7.07 (s,1H), 6.91 (m, 1H), 5.06 (d, J=7.3 Hz, 0.5H), 4.96 (m, 1H), 4.89 (d,J=5.2 Hz, 0.5H), 4.82 (d, J=7.5 Hz, 0.5H), 4.76 (d, J=4.2 Hz, 0.5H),4.45-4.38 (m, 1H), 4.21 (m, 1H), 4.01-3.86 (m, 2H), 3.55 (m, 1H), 3.40(dd, J=4.9, 12.6 Hz, 0.5H), 3.23 (m, 1.5H), 3.17-3.07 (m, 1H), 2.97-2.83(m, 2H).

Example 12

(±)-2-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[1-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(±)-2-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (1:1dichloromethane:dimethylformamide; reaction mixture stirred for 3 d;combined organic phases washed with 2 N NaOH, dried over Na₂SO₄).

mp 100-101° C. (dec.); CIMS m/e 462.2/464.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.33 (m, 1H), 8.41 (dd, J=2.8, 8.2 Hz, 1H),7.27-7.18 (m, 4H), 7.13 (m, 1H), 6.93 (d, J=5.8 Hz, 1H), 6.69 (d, J=0.8Hz, 1H), 4.98-4.75 (m, 3H), 3.99 (m, 0.5H), 3.93 (m, 0.5H), 3.81 (m,1.5H), 3.41-3.21 (m, 2.5H), 3.12 (m, 1H), 3.00-2.82 (m, 2H).

Example 12a

2-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid

2-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester(Krutosikova, A.; Kovac, J.; Dandarova, M.; Lesko, J.; Ferik, S.,Collect. Czech. Chem. Commun., 46: 2564-2573 (1981)) was hydrolyzedaccording to Procedure E (4 equiv 2 N NaOH, ethanol; reflux 5 h, roomtemperature overnight; after concentration to remove ethanol, residuepartitioned between ethyl acetate and 2 N HCl; combined organic phasesdried over Na₂SO₄; no purification).

¹H NMR (DMSO-d₆) δ 12.47 (br s, 1H), 11.67 (s, 1H), 6.76 (d, J=0.8 Hz,1H), 6.67 (t, J=0.8 Hz, 1H).

Example 13

2-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onehydrochloride were coupled according to Procedure A (2:1dichloromethane:dimethylformamide; reaction mixture stirred for 3 d;combined organic phases washed with 2 N NaOH, dried over Na₂SO₄).

mp 112-123° C. (dec.); CIMS m/e 492.1/494.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.31 (s, 0.5H), 11.27 (s, 0.5H), 7.71 (d, J=8.7 Hz,1H), 7.25-7.18 (m, 4H), 7.11 (m, 1H), 6.81 (s, 1H), 6.68 (d, J=2.9 Hz,1H), 5.05-4.73 (m, 3H), 4.44-4.34 (m, 1H), 4.17 (brs, 1H), 3.98-3.85 (m,2H), 3.56-3.48 (m, 1H), 3.40-3.06 (m, 3H), 2.94-2.80 (m, 2H).

Example 14

6H-Thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

6H-Thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 15:1dichloromethane:dimethylformamide; reaction mixture stirred for 3 d;after saturated aqueous NaHCO₃, combined organic phases washed withwater, dried over Na₂SO₄).

mp 179-184° C.; CIMS m/e 400.1 (MH⁺), 398.2 ((M−H)⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 11.79 (br s 1H), 8.52 (d, J=8.3 Hz, 1H),7.34-7.16 (m, 6H), 7.04 (m, 2H), 5.04 (d, J=5.1 Hz, 0.5H), 4.96 (d,J=4.9 Hz, 0.5H), 4.90-4.80 (m, 2H), 4.09-3.98 (m, 1H), 3.89 (m, 1.5H),3.49-3.29 (m, 2.5H), 3.19 (m, 1H), 3.08-2.91 (m, 2H).

Example 15

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide; reaction mixture stirred for 3 d;combined organic phases washed with water prior to saturated aqueousNaHCO₃, dried over Na₂SO₄).

mp 140-143° C.; CIMS m/e 476.1/478.0 ((M−H)⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 11.73 (m, 1H), 8.61 (d, J=8.3 Hz, 1H),7.34-7.15 (m, 6H), 5.05-4.84 (m, 3H), 4.15-3.85 (m, 2.5H), 3.48-2.95 (m,5.5H).

Example 16

2-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid1(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide; reaction mixture stirred for 3 d;combined organic phases washed with water prior to saturated aqueousNaHCO₃, dried over Na₂SO₄).

mp 128-130° C.; CIMS m/e 412.2 ((M−H)⁺), 414.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.40 (m, 1H), 8.38 (m, 1H), 7.37-7.05 (m, 6H), 6.65(s, 1H), 4.97 (d, J=5.2 Hz, 0.5H), 4.90-4.76 (m, 2.5H), 4.07-3.82 (m,2.5H), 3.42-3.25 (m, 2H), 3.13 (m, 1.5H), 3.01-2.87 (m, 2H), 2.44 (d,J=1 Hz, 3H).

Example 16a

2-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester

5-Methyl-2-thiophenecarboxaldehyde was annulated according to ProcedureH (acrylate organic phases dried over Na₂SO₄).

mp 129-130° C.; CIMS m/e 208.2 ((M−H)⁺), 210.2 (MH⁺).

¹H NMR (CDCl₃) δ 8.90 (br s, 1H), 7.04 (s, 1H), 6.63 (s, 1H), 4.33 (q,J=7.1 Hz, 1H), 2.54 (s, 3H), 1.36 (t, J=7.2 Hz, 3H).

Example 16b

2-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure D (after cooling to room temperature,acidification with 2 N HCl, extracted with ethyl acetate; organic phasesdried over Na₂SO₄; no purification).

CIMS m/e 180.2 ((M−H)⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 12.35 (br s, 1H), 11.72 (s, 1H), 6.95 (s,1H), 6.73 (s, 1H), 2.51 (s, 3H).

Example 17

2,4-Dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2,4-Dichloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.1 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 25:1dichloromethane:dimethylformamide; 2 d reaction time; combined organicphases washed with water prior to saturated aqueous NaHCO₃, dried overNa₂SO₄).

mp 203-204° C.; CIMS m/e 468.1/470.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.20 (s, 1H), 7.65-7.58 (m, 1H), 7.28-7.08 (m, 6H),5.04 (d, J=3.3 Hz, 1H), 4.98-4.80 (m, 3H), 4.08-3.95 (m, 1H), 3.91-3.74(m, 2H), 3.26-3.10 (m, 2H), 3.10-2.88 (m, 2H).

Example 18

2-Cyano-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide

2-Cyano-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-1-(3-hydroxy-azetidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B.

CIMS m/e 395.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.09 (s, 1H), 8.74 (d, J=8.5 Hz, 1H), 7.99 (s, 1H),7.29-7.12 (m, 6H), 5.68 (m, 1H), 4.58 (m, 1H), 4.41 (m, 1H), 4.28 (m,0.5H), 4.11-3.90 (m, 2H), 3.69 (m, 0.5H), 3.57-3.49 (m, 1H), 3.01-2.88(m, 2H).

Example 18a

2-Cyano-6H-thieno[2,3-b]pyrrole-5-carboxylic acid

2-Formyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid (Soth, S. et al.,Bull. Soc. Chim. Fr., 2511-2515 (1975)) was treated with hydroxylaminehydrochloride according to Procedure G (100° C. for 13 h, 125° C. for 7h; after cooling to room temperature, concentration gave crude product).

CIMS m/e 190.9 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 13.03 (br s, 1H), 12.39 (br s, 1H), 7.97 (s, 1H),7.04 (s, 1H).

Example 19

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-morpholin-4-yl-2-oxo-ethyl]-amide

2-chloro-6H-thieno[2,3-b]pyrrole-0.5-carboxylic acid and(2S)-amino-1-morpholin-4-yl-3-phenyl-propan-1-one hydrochloride (See,for example, Suzuki, K.; Fujita, H.; Sasaki, Y.; Shiratori, M.;Sakurada, S.; Kisara, K., Chem. Pharm. Bull., 36, 4834-40 (1988)) werecoupled according to Procedure C (solution of product in ethyl acetatewashed with water, dried over MgSO₄, concentrated).

mp 108-110° C.; CIMS m/e 416.3/418.2 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 11.84 (m, 1H), 8.65 (d, J=8.2 Hz, 1H) 7.39-7.13 (m,7H), 5.08 (q, J=7.6 Hz, 1H), 3.60-3.30 (m, 7H), 3.23 (m, 1H), 3.09-2.95(m, 2H).

Example 20

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-dimethylcarbamoyl-2-phenyl-ethyl]-amide

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-N,N-dimethyl-3-phenyl-propionamide trifluoroacetate (See, forexample, Holladay, M. et al., J. Med. Chem., 37: 630-5 (1994)) werecoupled according to Procedure C (product washed with ethyl acetate).

mp 234-235° C.; CIMS m/e 374.2/376.2 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 11.72 (s, 1H), 8.53 (d, J=8.1 Hz, 1H), 7.23 (m, 4H),7.13 (m, 3H), 5.01 (m, 1H), 3.01-2.88 (m, 5H), 2.78 (s, 3H).

Example 21

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(1,1-dioxo-1-thiazolidin-3-yl)-2-oxo-ethyl]-amide

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-1-(1,1-dioxo-1-thiazolidin-3-yl)-3-phenyl-propan-1-onehydrochloride (WO96/39384, Example 40a) were coupled according toProcedure C (solution of product in ethyl acetate washed with water,dried over MgSO₄, concentrated).

mp 125-129° C.; CIMS m/e 450.2/452.2 ((M−H)⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 11.80 (m 1H), 8.75 (dd, J=8.1, 12.9, 1H),7.36 (m, 2H), 7.26-7.14 (m, 5H), 5.08-4.97 (m, 1H), 4.81 (m, 0.5H), 4.63(d, J=11.4, 0.5H), 4.55 (d, J=12.5, 0.5H), 4.45 (d, J=12.4, 0.5H), 4.25(m, 1H), 3.90-3.75 (m, 1H), 3.55-3.35 (m, 2H), 3.05 (m, 2H).

Example 22

1-{(2S)-[(2-chloro-6H-thieno[2,3-b]pyrrole-5-carbonyl)-amino]-3-phenyl-propionyl}-piperidine-4-carboxylicacid ethyl ester

2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and1-[(2S)-amino-3-phenyl-propionyl]-piperidine-4-carboxylic acid ethylester hydrochloride were coupled according to Procedure C (solution ofproduct in ethyl acetate washed with water, dried over MgSO₄,concentrated).

mp 104-105° C.; CIMS m/e 486.2/488.2 ((M−H)⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 11.84 (m, 1H), 8.64 (t, J=8.8 Hz, 1H),7.32-7.14 (m, 7H), 5.10 (m, 1H), 4.30-3.89 (m, 4H), 3.15-2.92 (m, 3H),2.80-2.50 (m, 2H), 1.83-1.69 (m, 2H), 1.52-0.94 (m, 5H).

Example 22a

1-((2S)-tert-Butoxycarbonylamino-3-phenyl-propionyl)-piperidine-4-carboxylicacid ethyl ester

Boc-L-Phenylalanine (1.1 equiv) and piperidine-4-carboxylic acid ethylester were coupled according to Procedure A (1.5 equiv1-hydroxybenzotriazole hydrate, 1.3 equiv1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, roomtemperature, dichloromethane; reaction mixture poured into water,acidified with 1 N HCl; resultant precipitate filtered, filtrateextracted with CHCl₃; organic phase washed sequentially with water andbrine, dried over MgSO₄ before concentration).

CIMS m/e 405.2 (MH⁺).

¹H NMR (CDCl₃) δ 7.26-7.14 (m, 5H), 5.40 (dd, J=8.9, 19.3 Hz, 1H), 4.82(m, 1H), 4.34-4.24 (m, 1H), 4.09 (dq, J=2.0, 7.1 Hz, 2H), 3.57 (m, 1H),2.99-2.88 (m, 2.5H), 2.72 (m, 1H), 2.45-2.32 (m, 1.5H), 1.95-1.79 (m,1.5H), 1.58 (m, 2H), 1.40 (d, J=2.1 Hz, 9H), 1.23 (m, 3H), 0.68 (m,0.5H).

Example 22b

1-((2S)-Amino-3-phenyl-propionyl)-piperidine-4-carboxylic acid ethylester Hydrochloride

To a solution of1-((2S)-tert-butoxycarbonylamino-3-phenyl-propionyl)-piperidine-4-carboxylicacid ethyl ester (11 g, 27.20 mmol) in ethyl acetate (150 ml) wasbubbled in HCl gas over 10 min. The reaction mixture was stirredovernight, concentrated, redissolved in ethyl acetate and ether, andconcentrated. The crude product was precipitated with hexanes, filtered,and dried in vacuo to give the title product (9.1 mg, 98%).

CIMS m/e 305.1 (MH⁺).

¹H NMR (CDCl₃) δ 8.56 (br s, 2H), 7.29-7.18 (m, 5H), 4.94-4.82 (m, 1H),4.22-3.97 (m, 4H), 3.53 (dt, J=4.5, 12.7 Hz, 1H), 3.41-3.27 (m, 1H),3.12 (m, 1H), 2.95 (m, 0.5H), 2.76 (t, J=10.9 Hz, 0.5H), 2.66 (m, 0.5H),2.27 (m, 1H), 2.07 (m, 0.5H), 1.79-1.51 (m, 2H), 1.38-1.11 (m, 3.5H),0.41 (m, 0.5H).

Example 23

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide

2-Bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid and(2S)-amino-1-(3-hydroxy-azetidin-1-yl)-3-phenyl-propan-11-onehydrochloride were coupled according to Procedure B.

CIMS m/e 448.1/450.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.77 (s, 1H), 8.55 (d, J=8.1 Hz, 1H), 7.26-7.10 (m,7H), 5.00-4.76 (m, 3H), 4.07-3.94 (m, 1.5H), 3.83 (m, 1.5H), 3.40-3.22(m, 1H), 3.13 (m, 2H), 2.93 (m, 2H).

Example 24

2-Methyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Methyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid (Krutosikova, A.;Kovac, J.; Dandarova, M.; Lesko, J.; Ferik, S., Collect. Czech. Chem.Commun., 46: 2564-2573 (1981)) and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (acidic aqueousphase extracted with ethyl acetate; organic phases combined prior tobasic work-up).

CIMS m/e 396.3 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 10.96 (m, 1H), 8.23 (m, 1H), 7.27-7.19 (m, 4H), 7.14(m, 1H), 6.83 (d, J=5.4 Hz, 1H), 6.15 (s, 1H), 4.97 (d, J=5.2 Hz, 0.5H),4.89 (d, J=4.6 Hz, 0.5H), 4.80 (m, 2H), 3.99 (m, 0.5H), 3.93 (m, 0.5H),3.82 (m, 1.5H), 3.38 (m, 1H), 3.25 (m, 1H), 3.13 (m, 1.5H), 3.00-2.85(m, 2H), 2.31 (s, 3H).

Example 25

2-Trimethylsilanylethynyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide

Using a modified procedure of J. M. Tour et al. (J. Org. Chem., 61:6906-6921 (1996)), to a degassed solution of2-bromo-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide (106 mg,0.24 mmol) in tetrahydrofuran (5 ml) was sequentially addeddiisopropylamine (36 μl, 0.26 mmol), a mixture of copper(1) iodide (9mg, 0.05 mmol) and dichlorobis(triphenylphosphine)palladium(II) (68 mg,0.1 mmol), and (trimethylsilyl)acetylene (41 μl, 0.29 mmol). The mixturewas stirred overnight, poured into water, and extracted withdichloromethane. The combined organic phases were washed with saturatedNaCl, dried over MgSO₄, and concentrated. The product was purified byChromatotron-chromatography (dichloromethane; 20:1dichloromethane:methanol) to give the title product (4.5 mg, 4%).

CIMS m/e 464.3 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 11.86 (s, 1H), 8.54 (t, J=8.9 Hz, 1H), 7.31 (s, 1H),7.23 (m, 4H), 7.13 (m, 2H), 5.66 (m, 1H), 4.56 (m, 1H), 4.41 (m, 1H),4.28 (m, 0.5H), 4.11-3.89 (m, 2H), 3.66 (m, 0.5H), 3.57-3.46 (m, 1H),2.99-2.85 (m, 2H), 0.23 (s, 9H).

Example 26

2-Ethynyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide

Using a procedure analogous to that of G. M. Whitesides et al. (J. Org.Chem., 53: 2489-2496 (1988)), to a solution of2-trimethylsilanylethynyl-6H-thieno[2,3-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide (110 mg,0.02 mmol) in methanol (0.5 ml) was added a 5% aqueous solution ofpotassium hydroxide (7 μL, 0.06 mmol). The reaction mixture was stirredfor 3 h, concentrated to remove the methanol, diluted with water, andextracted with dichloromethane. The organic phase dried over MgSO₄ andconcentrated to give the title product (7 mg, 77%).

CIMS m/e 392.1 ((M−H)⁺).

¹H NMR (CDCl₃) δ 7.32-7.16 (m, 7H), 7.03 (m, 2H), 4.66 (m, 1H), 4.47 (m,1H), 4.17 (m, 1H), 4.00 (m, 1H), 3.75-3.56 (m, 3H), 3.35 (m, 1H), 3.04(m, 2H).

Example 27

2-Fluoro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Fluoro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (4 d reaction time).

mp 128-132° C.; CIMS m/e 416.1 ((M−H)⁺), 418.2 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.72 (m, 1H), 8.49 (d, J=8.5 Hz, 1H), 7.28-7.13 (m,6H), 6.71 (s, 1H), 4.99 (d, J=5.2 Hz, 0.5H), 4.91 (d, J=4.8 Hz, 0.5H),4.86 (d, J=3.7 Hz, 1H), 4.79 (m, 1H), 4.01 (m, 0.5H), 3.94 (m, 0.5H),3.83 (m, 1.5H), 3.42-3.24 (m, 2.5H), 3.14 (m, 1H), 3.01-2.84 (m, 2H).

Example 27a

2-Fluoro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester

5-Fluoro-thiophene-2-carbaldehyde (see, for example, Schuetz, R. D. andNilles, G. P., J. Org. Chem., 36: 2188-90 (1971)) was annulatedaccording to Procedure H (aldehyde and azido-acetic acid ethyl esteradded as ethanol solution (0.6 M of ester); acrylate organic phasewashed with saturated aqueous NaCl prior to drying; acrylate notpurified).

CIMS m/e 212.1 ((M−H)⁺).

¹H NMR (CDCl₃) δ 9.16 (br s, 1H), 7.03 (s, 1H), 6.51 (s, 1H), 4.33 (q,J=7.2 Hz, 2H), 1.36 (t, J=7.2 Hz, 3H).

Example 27b

2-Fluoro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-Fluoro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure F (acidified aqueous phase extractedwith ethyl acetate; combined organic phases dried over MgSO₄,concentrated).

CIMS m/e 184.1 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.47 (brs, 1H), 12.03 (s, 1H), 6.96 (s, 1H), 6.73(s, 1H).

Example 28

2-Cyano-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide

2-Cyano-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-(3-hydroxy-azetidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (reaction mixturepartitioned between ethyl acetate and water prior to acidic washing).

CIMS m/e 377.1 ((M−H)⁺), 379.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.78 (s, 1H), 8.68 (t, J=8.2 Hz, 1H), 7.67 (s, 1H),7.22 (m, 4H), 7.15 (m, 1H), 7.01 (d, J=3.1 Hz, 1H), 5.68 (m, 1H), 4.59(m, 1H), 4.40 (m, 1H), 4.26 (m, 0.5H), 4.05 (m, 1H), 3.92 (m, 1H), 3.65(m, 0.5H), 3.53 (m, 1H), 3.00-2.88 (m, 2H).

Example 28a

2-Cyano-4H-furo[3,2-b]pyrrole-5-carboxylic acid

2-Formyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid (see, for example,Krutosikova, A.; Dandarova, M.; Alfoldi, J., Chem. Pap., 48: 268-73(1994)) was treated with hydroxylamine hydrochloride according toProcedure G.

CIMS m/e 174.9 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 13.10-12.60 (brs, 1H), 12.05 (s, 1H), 7.73 (s, 1H),6.75 (s, 1H).

Example 29

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-11-onehydrochloride were coupled according to Procedure B (3 d reaction time;reaction mixture partitioned between ethyl acetate and water prior toacidic washing).

CIMS m/e 418.1/420.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.36 (s, 1H), 8.42 (dd, J=2.9, 8.3 Hz, 1H),7.27-7.10 (m, 5H), 6.94 (d, J=6.0 Hz, 1H), 6.63 (m, 1H), 4.99 (d, J=5.2Hz, 0.5H), 4.91 (d, J=5.0 Hz, 0.5H), 4.86-4.77 (m, 2H), 4.00 (m, 0.5H),3.94 (m, 0.5H), 3.81 (m, 1.5H), 3.43-3.21 (m, 2.5H), 3.13 (m, 1H),3.00-2.85 (m, 2H).

Example 29a

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester

5-chloro-furan-2-carbaldehyde (Snyder, H. R., Jr.; Seehausen, P. H., J.Heterocycl. Chem., 10: 385-6 (1973)) was annulated according toProcedure H (8 equiv sodium; aldehyde and azido-acetic acid ethyl esteradded as ethanol solution (0.9 M of ester); condensation reactionmixture allowed to warm to room temperature, stirred for 1 h, quenchedat −40° C., diluted with water, and extracted with ether; acrylate notpurified; crude furanopyrrole filtered before concentration).

CIMS m/e 212.0/214.1 ((M−H)⁺).

¹H NMR (CDCl₃) δ 8.69 (br s, 1H), 6.74 (dd, J=0.8, 1.7 Hz, 1H), 6.31 (d,J=0.6 Hz, 1H), 4.33 (q, J=7.1 Hz, 2H), 1.36 (t, J=7.1 Hz, 3H).

Example 29b

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure F (room temperature overnight, 50° C.4 h; acidified aqueous phase extracted with ethyl acetate; combinedorganic phases dried over MgSO₄, concentrated).

CIMS m/e 183.8/185.8 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.47 (br s, 1H), 11.70 (s, 1H), 6.70 (s, 1H), 6.67(s, 1H).

Example 30

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-chloro-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure B (3 d reaction time; reactionmixture partitioned between ethyl acetate and water prior to acidicwashing).

CIMS m/e 448.1/450.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.33 (m, 1H), 7.72 (d, J=8.7 Hz, 1H), 7.25-7.09 (m,5H), 6.82 (s, 1H), 6.61 (dd, J=0.7, 3.0 Hz, 1H), 5.04 (d, J=7.3 Hz,0.5H), 4.94 (m, 1H), 4.89 (d, J=5.0 Hz, 0.5H), 4.80 (d, J=7.5 Hz, 0.5H),4.75 (d, J=4.2 Hz, 0.5H), 4.45-4.35 (m, 1H), 4.18 (m, 1H), 4.00-3.88 (m,2H), 3.57-3.49 (m, 1H), 3.39 (m, 0.5H), 3.26-3.06 (m, 2.5H), 2.95-2.80(m, 2H).

Example 31

1-{(2S)-[(2-chloro-6H-thieno[2,3-b]pyrrole-5-carbonyl)-amino]-3-phenyl-propionyl}-piperidine-4-carboxylicacid

1-{(2S)-[(2-chloro-6H-thieno[2,3-b]pyrrole-5-carbonyl)-amino]-3-phenyl-propionyl}-piperidine-4-carboxylicacid ethyl ester was hydrolyzed according to Procedure F (roomtemperature; after concentration, reaction residue partitioned betweenethyl acetate and 1-2 N NaOH; aqueous phase acidified with 2 N HCl,extracted with ethyl acetate; combined organic phases dried over MgSO₄,concentrated; crude product washed with ether).

mp 145-150° C.

¹H NMR (DMSO-d₆) δ 12.21 (s, 1H), 11.83 (s, 0.5H), 11.77 (s, 0.5H), 8.58(m, 1H), 7.26-7.11 (m, 7H), 5.05 (m, 1H), 4.23 (d, J=13.3 Hz, 0.5H),4.10 (d, J=12.5 Hz, 0.5H), 3.93 (d, J=12.7 Hz, 0.5H), 3.85 (d, J=13.5Hz, 0.5H), 3.11-2.90 (m, 3H), 2.77-2.61 (m, 1H), 2.49-2.39 (m, 1H),1.75-1.65 (m, 2H), 1.43-1.17 (m, 1.5H), 1.07-0.97 (m, 0.5H).

Example 32

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (reaction mixturepartitioned between ethyl acetate and water prior to acidic washing).

CIMS m/e 434.0/436.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.09 (m, 1H), 8.57 (d, J=8.3 Hz, 1H), 7.40 (m,0.5H), 7.28-7.10 (m, 6.5H), 5.00 (d, J=5.2 Hz, 0.5H), 4.92 (d, J=5.0 Hz,0.5H), 4.84 (m, 2H), 4.10-3.93 (m, 1H), 3.82 (m, 1.5H), 3.44-3.23 (m,2.5H), 3.13 (m, 1H), 3.03-2.87 (m, 2H).

Example 32a

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester

4-chloro-thiophene-2-carbaldehyde (Iriarte, J.; Martinez, E.; Muchowski,J. M., J. Heterocycl. Chem., 13: 393-4 (1976)) was annulated accordingto Procedure H (aldehyde and azido-acetic acid ethyl ester added asethanol solution (1.2 M of ester) such that reaction temperaturemaintained at 0° C.; reaction mixture allowed to warm to 10° C., stirredfor 1.5 h, poured into cold saturated aqueous NH₄Cl; after etherextractions, combined acrylate organic phases washed with water untilaqueous phase was neutral; acrylate not purified).

CIMS m/e 228.0 ((M−H)⁺).

¹H NMR (CDCl₃) δ 9.02 (br s, 1H), 7.24 (s, 1H), 7.10 (s, 1H), 4.37 (q,J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H).

Example 32b

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure F (7 equiv LiOH—H₂O; room temperatureovernight, then at 50° C. overnight again; acidified aqueous phaseextracted with ethyl acetate; combined organic phases dried over MgSO₄,concentrated).

CIMS m/e 199.9/201.8 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.71 (br s, 1H), 12.40 (s, 1H), 7.48 (s, 1H), 7.06(d, J=1.9 Hz, 1H).

Example 33

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

3-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure B (reaction mixture partitionedbetween ethyl acetate and water prior to acidic washing).

CIMS m/e 464.0/466.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.4 (m, 1H), 7.89 (d, J=8.9 Hz, 1H), 7.38 (s, 0.5H),7.26-7.10 (m, 5.5H), 7.05 (d, J=3.1 Hz, 1H), 5.08 (d, J=7.1 Hz, 0.5H),4.97-4.84 (m, 2H), 4.76 (d, J=4.2 Hz, 0.5H), 4.44 (m, 1H), 4.20 (m, 1H),4.09-3.88 (m, 2H), 3.53 (m, 1H), 3.38 (m, 0.5H), 3.25-3.06 (m, 2.5H),2.97-2.82 (m, 2H).

Example 34

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (reaction mixturepartitioned between ethyl acetate and water prior to acidic washing).

CIMS m/e 475.9/478.2 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 11.99 (m, 1H), 8.56 (d, J=8.3 Hz, 1H), 7.48 (d, J=1.2Hz, 0.5H), 7.27-7.12 (m, 6.5H), 4.99 (d, J=5.2 Hz, 0.5H), 4.91 (d, J=5.2Hz, 0.5H), 4.84 (m, 2H), 4.09-3.92 (m, 1.5H), 3.78 (m, 1.5H), 3.43-3.22(m, 2H), 3.13 (m, 1H), 2.99-2.85 (m, 2H).

Example 34a

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

4-Bromo-thiophene-2-carbaldehyde was annulated according to Procedure H(aldehyde and azido-acetic acid ethyl ester added as ethanol solution(1.2 M of ester) such that reaction temperature maintained at 0° C.;reaction mixture allowed to warm to 10° C., stirred for 1 h, poured intocold saturated aqueous NH₄Cl; after ether extractions, combined acrylateorganic phases washed with water until aqueous phase was neutral;acrylate not purified).

CIMS m/e 272.0/273.9 ((M−H)⁺).

¹H NMR (CDCl₃) δ 8.99 (br s, 1H), 7.21 (s, 1H), 7.13 (d, J=1.9 Hz, 1H),4.37 (q, J=7.2 Hz, 2H), 1.38 (t, J=7.2 Hz, 3H).

Example 34b

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure F (7 equiv LiOH—H₂O; room temperatureovernight, then at 50° C. overnight again; acidified aqueous phaseextracted with ethyl acetate; combined organic phases dried over MgSO₄,concentrated).

CIMS m/e 243.9/245.9 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.69 (br s, 1H), 12.33 (s, 1H), 7.56 (s, 1H), 7.08(s, 1H).

Example 35

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure B (reaction mixture partitionedbetween ethyl acetate and water prior to acidic washing).

CIMS m/e 508.0/510.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.96 (s, 0.5H), 11.91 (s, 0.5H), 7.90 (d, J=9.3 Hz,1H), 7.46 (s, 0.5H), 7.22 (m, 4.5H), 7.12 (m, 1H), 7.04 (m, 1H), 5.08(d, J=6.9 Hz, 0.5H), 4.94 (m, 1H), 4.89 (d, J=5.0 Hz, 0.5H), 4.85 (d,J=7.1 Hz, 0.5H), 4.75 (d, J=4.4 Hz, 0.5H), 4.45 (m, 1H), 4.20 (m, 1H),4.08-3.87 (m, 2H), 3.53 (m, 1H), 3.40-3.30 (m, 0.5H), 3.22 (m, 1H),3.15-3.05 (m, 1.5H), 2.98-2.82 (m, 2H).

Example 36

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure B (reaction mixture partitionedbetween ethyl acetate and water prior to acidic washing).

CIMS m/e 464.0/466.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.72 (s, 0.5H), 11.67 (s, 0.5H), 7.85 (d, J=9.1 Hz,1H), 7.21 (m, 4H), 7.12 (m, 1H), 7.00 (m, 2H), 5.05 (d, J=7.1 Hz, 0.5H),4.95 (m, 1H), 4.89 (d, J=4.8 Hz, 0.5H), 4.81 (d, J=7.5 Hz, 0.5H), 4.75(d, J=3.9 Hz, 0.5H), 4.41 (m, 1H), 4.20 (m, 1H), 4.00-3.87 (m, 2H), 3.54(m, 1H), 3.40 (m 0.5H), 3.22 (m, 1.5H), 3.15-3.06 (m, 1H), 2.94-2.80 (m,2H).

Example 36a

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester

5-chloro-thiophene-2-carbaldehyde was annulated according to Procedure H(aldehyde and azido-acetic acid ethyl ester added as ethanol solution(1.2 M of ester) such that reaction temperature maintained at 0-5° C.;reaction mixture allowed to warm to room temperature, stirred for 2 h,poured into cold saturated aqueous NH₄Cl; after ether extractions,combined acrylate organic phases washed with water until aqueous phasewas neutral; 0.5 M solution of crude acrylate heated for 1.5 h).

CIMS m/e 228.0/229.9 ((M−H)⁺).

¹H NMR (CDCl₃) δ 9.04 (br s, 1H), 7.02 (m, 1H), 6.88 (m, 1H), 4.34 (q,J=7.2 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

Example 36b

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure F (50° C. 9 h).

CIMS m/e 199.9/201.8 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.62 (s, 1H), 12.04 (s, 1H), 7.05 (s, 1H), 6.97 (s,1H).

Example 37

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (reaction mixturepartitioned between ethyl acetate and water prior to acidic washing).

CIMS m/e 434.0/436.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.73 (m, 1H), 8.57 (d, J=7.9 Hz, 1H), 7.28-7.19 (m,4H), 7.13 (m, 2H), 7.01 (d, J=2.7 Hz, 1H), 5.00 (d, J=5.2 Hz, 0.5H),4.92 (d, J=5.2 Hz, 0.5H), 4.86 (m, 1H), 4.79 (m, 1H), 4.08-3.94 (m, 1H),3.82 (m, 1.5H), 3.42-3.24 (m, 2H), 3.14 (m, 1.5H), 3.01-2.87 (m, 2H).

Example 38

3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (reaction mixturepartitioned between ethyl acetate and water prior to acidic washing).

CIMS m/e 412.1 ((M−H)⁺), 414.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.69 (m, 1H), 8.45 (m, 1H), 7.28-7.18 (m, 4H), 7.12(m, 2H), 6.93 (d, J=1.0 Hz, 1H), 4.98 (d, J=5.2 Hz, 0.5H), 4.90 (d,J=5.0 Hz, 0.5H), 4.83 (m, 2H), 4.03-3.92 (m, 1H), 3.83 (m, 1.5H),3.42-3.23 (m, 2.5H), 3.14 (m, 1H), 3.03-2.88 (m, 2H), 2.24 (s, 3H).

Example 38a

3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester

4-Methyl-thiophene-2-carbaldehyde (Detty, M. R.; Hays, D. S.,Heterocycles, 40: 925-37 (1995)) was annulated according to Procedure H(aldehyde and azido-acetic acid ethyl ester added as ethanol solution(1.1 M of ester); reaction poured into cold saturated aqueous NH₄Cl;after ether extractions, acrylate organic phase washed with water untilaqueous phase was neutral; acrylate not purified).

CIMS m/e 207.9 ((M−H)⁺), 209.9 (MH⁺).

¹H NMR (CDCl₃) δ 9.02 (br s, 1H), 7.09 (d, J=1.9 Hz, 1H), 6.91 (d, J=1.0Hz, 1H), 4.35 (quart, J=7.3 Hz, 1H), 2.32 (d, J=1.2 Hz, 3H), 1.38 (t,J=7.2 Hz, 3H).

Example 38b

3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure F (50° C. 13 h; acidified aqueousphase extracted with ethyl acetate; combined organic phases dried overMgSO₄, concentrated).

CIMS m/e 179.9 ((M−H)⁺), 181.8 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.45 (br s, 1H), 11.99 (s, 1H), 7.02 (m, 1H), 6.96(m, 1H), 2.25 (s, 3H).

Example 39

3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure B (reaction mixture partitionedbetween ethyl acetate and water prior to acidic washing).

CIMS m/e 442.1 ((M−H)⁺), 444.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.66 (s, 0.5H), 11.62 (s, 0.5H), 7.76 (d, J=8.9 Hz,1H), 7.22 (m, 4H), 7.11 (m, 1H), 7.01 (d, J=1.7 Hz, 1H), 6.91 (s, 1H),5.06 (d, J=7.3 Hz, 0.5H), 4.95 (m, 1H), 4.90 (d, J=5.0 Hz, 0.5H), 4.82(d, J=7.5 Hz, 0.5H), 4.77 (d, J=4.4 Hz, 0.5H), 4.44 (m, 1H), 4.21 (m,1H), 4.01-3.87 (m, 2H), 3.55 (m, 1H), 3.40 (dd, J=5.0, 12.6 Hz, 0.5H),3.24 (m, 1.5H), 3.16-3.06 (m, 1H), 2.97-2.83 (m, 2H), 2.23 (s, 3H).

Example 40

2-Cyano-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Cyano-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (reaction mixturepartitioned between ethyl acetate and water prior to acidic washing).

CIMS m/e 423.1 ((M−H)⁺), 425.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.15 (m, 1H), 8.85 (d, J=8.3 Hz, 1H), 7.79 (m, 1H),7.29-7.11 (m, 6H), 5.00 (m, 0.5H), 4.93-4.78 (m, 2.5H), 4.04-3.93 (m,1H), 3.81 (m, 1.5H), 3.43-3.25 (m, 2.5H), 3.15 (m, 1H), 3.03-2.89 (m,2H).

Example 40a

2-Formyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-Formyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (see, forexample, Gale, W. W. et al., J. Org. Chem., 29: 2160-2165 (1964)) washydrolyzed according to Procedure F (50° C. overnight; acidified aqueousphase extracted with ethyl acetate; combined organic phases dried overMgSO₄, concentrated).

CIMS m/e 193.9 ((M−H)⁺), 195.8 (MH⁺).

¹H NMR (DMSO-d₆) δ 13.38-12.78 (br s, 1H), 12.43 (s, 1H), 9.90 (s, 1H),7.92 (s, 1H), 7.08 (s, 1H).

Example 40b

2-Cyano-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-Formyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid was treated withhydroxylamine hydrochloride according to Procedure G.

CIMS m/e 190.9 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 13.06 (br s, 1H), 12.45 (s, 1H), 7.84 (s, 1H), 7.09(s, 1H).

Example 41

2-Cyano-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-Cyano-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure A (1 equiv triethylamine,dimethylformamide; 4 d reaction time; reaction mixture partitionedbetween ethyl acetate and water; organic phase washed with 2 N HCl priorto saturated aqueous NaHCO₃).

mp 137-140° C.; CIMS m/e 437.1 ((M−H)⁺), 439.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.70 (m, 1H), 7.99 (d, J=8.9 Hz, 1H), 7.65 (m, 1H),7.22 (m, 4H), 7.12 (m, 1H), 6.91 (s, 1H), 5.09 (d, J=7.1 Hz, 0.5H), 4.95(m, 1H), 4.89 (d, J=5.2 Hz, 0.5H), 4.83 (d, J=7.3 Hz, 0.5H), 4.76 (d,J=3.9 Hz, 0.5H), 4.42 (m, 1H), 4.21 (m, 1H), 4.08-3.89 (m, 2H),3.61-3.50 (m, 1H), 3.40 (m, 0.5H), 3.24 (m, 1.5H), 3.13 (m, 1H),2.95-2.80 (m, 2H).

Example 42

3-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

3-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure A (dimethylformamide;reaction mixture partitioned between ethyl acetate and water; organicphase washed with 2 N HCl prior to saturated aqueous NaHCO₃).

mp 140° C. dec.; CIMS m/e 461.9/463.9 (MH⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 11.75 (m, 1H), 8.47 (d, J=8.6 Hz, 1H), 7.90(d, J=0.8 Hz, 1H), 7.33-7.15 (m, 5H), 6.98 (dd, J=1.5, 3.4 Hz, 1H), 5.03(d, J=5.3 Hz, 0.5H), 4.95 (d, J=5.1 Hz, 0.5H), 4.91-4.83 (m, 2H),4.13-3.97 (m, 1H), 3.86 (m, 1.5H), 3.48-3.25 (m, 2.5H), 3.18 (m, 1H),3.08-2.92 (m, 2H).

Example 42a

3-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester

4-Bromo-2-furaldehyde was annulated according to Procedure H (aldehydeand azido-acetic acid ethyl ester added as ethanol solution (1 M ofester) to −20° C. ethoxide solution; −20° C. 35 min, −5° C. 1.5 h, 5° C.15 min; reaction poured into cold saturated aqueous NH₄Cl; after etherextraction, acrylate organic phase washed with water until aqueous phasewas neutral; 0.5 M solution of crude acrylate heated).

CIMS m/e 257.8/259.8 (MH⁺).

¹H NMR (300 MHz, CDCl₃) δ 8.81 (br s, 1H), 7.48 (s, 1H), 6.79 (d, J=1.8Hz, 1H), 4.35 (q, J=7.1 Hz, 2H), 1.36 (t, J=7.1 Hz, 3H).

Example 42b

3-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid

3-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester washydrolyzed according to Procedure F (50° C. 14 h; acidified aqueousphase extracted with ethyl acetate; organic phase dried over MgSO₄,concentrated).

CIMS m/e 228.0/230.0 ((M−H)⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 12.60 (br s, 1H), 12.06 (br s, 1H), 7.98 (s,1H), 6.77 (d, J=1.8 Hz, 1H).

Example 43

3-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

3-Bromo-4H-furo[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure A (1 equiv triethylamine,dimethylformamide; reaction mixture partitioned between ethyl acetateand water; organic phase washed with 2 N HCl prior to saturated aqueousNaHCO₃).

mp 140° C. dec; CIMS m/e 492.0/494.0 (MH⁺).

¹H NMR (300 MHz, DMSO-d₆) δ 11.73 (s, 0.5H), 11.68 (s, 0.5H), 7.88 (d,J=0.7 Hz, 1H), 7.79 (d, J=8.9 Hz, 1H), 7.27 (m, 4H), 7.17 (m, 1H), 6.87(s, 1H), 5.18-4.74 (m, 3H), 4.56-4.40 (m, 1H), 4.24 (s, 1H), 4.05-3.94(m, 1.5H), 3.64-3.11 (m, 4.5H), 3.02-2.85 (m, 2H).

Example 44

4H-1,7-Dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

4H-1,7-Dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure B (2:1dichloromethane:dimethylformamide; reaction mixture partitioned betweenethyl acetate and water; organic phase washed with 2 N HCl prior tosaturated aqueous NaHCO₃).

CIMS m/e 484.0 ((M−H)⁺), 486.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.00 (s, 0.5H), 11.95 (s, 0.5H), 7.83 (m, 1H), 7.55(dd, J=0.8, 5.2 Hz, 1H), 7.35 (dd, J=1.2, 5.2 Hz, 1H), 7.23 (m, 4H),7.12 (m, 2H), 5.07 (d, J=7.3 Hz, 0.5H), 4.96 (m, 1H), 4.90 (d, J=5.0 Hz,0.5H), 4.81 (d, J=7.5 Hz, 0.5H), 4.76 (d, J=4.2 Hz, 0.5H), 4.44 (m, 1H),4.20 (m, 1H), 4.08-3.88 (m, 1.5H), 3.57 (m, 1H), 3.40 (m, 0.5H), 3.26(m, 1.5H), 3.17-3.07 (m, 1.5H), 2.97-2.84 (m, 2H).

Example 44a

4H-1,7-Dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid ethyl ester

Thieno[2,3-b]thiophene-2-carbaldehyde (Dopper, J. H. et al., J. Am.Chem. Soc., 95: 3692-8 (1973)) was annulated according to Procedure H(aldehyde and azido-acetic acid ethyl ester added as ethanol solution (1M of ester) to −20° C. ethoxide solution; −20° C. 30 min, −20° C. toroom temperature over 2.5 h; reaction poured into cold saturated aqueousNH₄Cl; after ether extraction, acrylate organic phase washed with wateruntil aqueous phase was neutral; 0.35 M solution of crude acrylateheated).

CIMS m/e 250.1 ((M−H)⁺), 251.9 (MH⁺).

¹H NMR (CDCl₃) δ 9.46 (br s, 1H), 7.35 (d, J=5.3 Hz, 1H), 7.29 (d, J=5.3Hz, 1H), 7.14 (d, J=2.0 Hz, 1H), 4.38 (q, J=7.1 Hz, 2H), 1.39 (t, J=7.1Hz, 3H).

Example 44b

4H-1,7-Dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid

4H-1,7-Dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid ethyl esterwas hydrolyzed according to Procedure F (50° C. 11 h).

CIMS m/e 222.0 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.51 (s, 1H), 12.32 (s, 1H), 7.59 (d, J=5.3 Hz, 1H),7.38 (d, J=5.3 Hz, 1H), 7.05 (d, J=1.9 Hz, 1H).

Example 45

4H-1,7-Dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

4H-1,7-Dithia-4-aza-cyclopenta[a]pentalene-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (1:1dichloromethane:dimethylformamide; reaction mixture partitioned betweenethyl acetate and water; organic phase washed with 2 N HCl prior tosaturated aqueous NaHCO₃).

CIMS m/e 454.0 ((M−H)⁺), 456.0 (MH⁺).

¹H NMR (DMSO-d₆) δ 12.03 (m, 1H), 8.53 (d, J=8.1 Hz, 1H), 7.55 (dd,J=0.8, 5.2 Hz, 1H), 7.34 (dd, J=2.7, 5.2 Hz, 1H), 7.29-7.19 (m, 5H),7.12 (m, 1H), 4.99 (d, J=5.2 Hz, 0.5H), 4.91 (d, J=5.0 Hz, 0.5H), 4.84(m, 2H), 3.98 (m, 1H), 3.83 (m, 2H), 3.41-3.29 (m, 3H), 3.13 (m, 1H),2.95 (m, 2H).

Example 46

2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (1:1dichloromethane:dimethylformamide; 2 d reaction time; reaction mixtureconcentrated to remove dichloromethane; partitioned between ethylacetate and water; organic phase washed with 2 N HCl prior to saturatedaqueous NaHCO₃).

mp 139-141° C.; CIMS m/e 448.1/450.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.95 (m, 1H), 8.54 (d, J=7.9 Hz, 1H), 7.28-7.18 (m,4H), 7.13 (m, 2H), 4.98 (d, J=5.2 Hz, 0.5H), 4.90 (d, J=4.6 Hz, 0.5H),4.83 (m, 2H), 4.02-3.92 (m, 1H), 3.82 (m, 1.5H), 3.41-3.22 (m, 2.5H),3.14 (m, 1H), 3.01-2.90 (m, 2H), 2.20 (d, J=2.5 Hz, 3H).

Example 46a

5-chloro-4-methyl-thiophene-2-carbaldehyde

Using a modified procedure of Silverstein et al. (Organic SynthesisColl. Vol 4, Wiley, New York, 1963, N. Rabjohn, ed. p 831), to a 80° C.pale yellow solution of 2-chloro-3-methyl-thiophene (Crast, L. B., Jr.U.S. Pat. No. 3,290,291, Example 2; 70 g, 0.53 mol) in dimethylformamide(48.3 g, 0.66 mol) was added phosphorous oxychloride (101.5 g, 0.66 mol)dropwise over 45 min, while maintaining temperature at 80-97° C. Thedark brown solution was stirred at 90° C. for 3 h and poured slowly intowater (500 mL) at 90° C. The resultant mixture was steam distilled andthe distillate was cooled to 0° C., affording white crystals. The first500 ml of distillate was extracted with chloroform and concentrated andthe residue was recrystallized from hexane (150 ml) at −50° C. The crudeproduct (8.6 g) was dissolved in hexane (100 ml) and the insolublematerial was filtered. The filtrate was diluted with hexane (50 ml),stirred with Norit® (2 g), and concentrated. The product was purified byrecrystallization from hexane (50 ml) at −40° C. and obtained as whitecrystals (7.5 g, 13%). The remaining distillate from the steamdistillation was extracted with chloroform (2×250 ml) and the whitecrystals dissolved in chloroform (1.5 l). The combined organic phaseswere dried over MgSO₄, filtered, stirred with Norit® (30 g) for 15 min,and concentrated. The residue was dissolved in hexane (350 ml), theinsoluble material was filtered, the filtrate was stirred at −15° C. for10 min, and the resultant precipitate was filtered. The title productwas obtained as pale yellow crystals (48.6 g, 83%).

mp 39-40° C.

Example 46b

2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester

5-chloro-4-methyl-thiophene-2-carbaldehyde was annulated according toProcedure H (aldehyde and azido-acetic acid ethyl ester was added asethanol solution (1 M of ester) to −20° C. ethoxide solution; allow towarm to 10° C. over 2 h, 10° C. 2 h; reaction poured into cold saturatedaqueous NH₄Cl; after ether extraction, acrylate organic phase washedwith water until aqueous phase was neutral; solution of crude acrylateadded to refluxing xylenes over 5 min and then heated at reflux).

CIMS m/e 243.8/245.9 (MH⁺).

¹H NMR (CDCl₃) δ 9.25 (br s, 1H), 7.02 (d, J=1.9 Hz, 1H), 4.36 (q, J=7.1Hz, 2H), 2.28 (s, 3H), 1.38 (t, J=7.1 Hz, 3H).

Example 46c

2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl esterwas hydrolyzed according to Procedure F (50° C. 14 h).

CIMS m/e 213.8/215.8 ((M−H)⁺).

¹H NMR (DMSO-d₆) δ 12.61 (br s, 1H), 12.25 (s, 1H), 6.96 (dd, J=0.5, 2.0Hz, 1H), 2.23 (s, 1H).

Example 47

2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide

2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(3S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-4-phenyl-butan-1-onewere coupled according to Procedure B (4:5dichloromethane:dimethylformamide; 2 d reaction time; reaction mixtureconcentrated to remove dichloromethane; partitioned between ethylacetate and water; organic phase washed with 2 N HCl prior to saturatedaqueous NaHCO₃).

mp 150-153° C.; CIMS m/e 478.1/480.1 (MH⁺).

¹H NMR (DMSO-d₆) δ 11.91 (s, 0.5H), 11.86 (s, 0.5H), 7.82 (d, J=8.7,1H), 7.22 (m, 4H), 7.11 (m, 1H), 7.01 (m, 1H), 5.07 (d, J=6.8 Hz, 0.5H),4.95 (m, 1H), 4.90 (d, J=5.0 Hz, 0.5H), 4.82 (d, J=6.8 Hz, 0.5H), 4.77(d, J=3.7 Hz, 0.5H), 4.44 (m, 1H), 4.21 (m, 1H), 4.08-3.88 (m, 1.5H),3.56 (m, 1H), 3.40 (m, 0.5H), 3.24 (m, 1.5H), 3.14 (m, 1H), 3.08 (m,0.5H), 2.95-2.82 (m, 2H).

Example 48

2-Methylsulfanyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide

2-Methylsulfanyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and(2S)-amino-1-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-3-phenyl-propan-1-onehydrochloride were coupled according to Procedure B (1:1dichloromethane:dimethylformamide; reaction mixture concentrated toremove dichloromethane, partitioned between ethyl acetate and water;organic phase washed with 2 N HCl prior to saturated aqueous NaHCO₃).

mp 104-110° C.; 444.0 ((M−H)⁺), 445.9 (MH⁺).

¹H NMR (DMSO-d₆)) δ 11.61 (m, 1H), 8.52 (d, J=8.6 Hz, 1H), 7.28-7.12 (m,6H), 6.97 (d, J=3.9 Hz, 1H), 4.99 (d, J=5.1 Hz, 0.5H), 4.91 (d, J=5.1Hz, 0.5H), 4.83 (m, 2H), 4.06-3.94 (m, 1H), 3.80 (m, 2H), 3.43-3.24 (m,2H), 3.14 (m, 1H), 3.02-2.87 (m, 2H), 2.46 (s, 3H).

Example 48a

2-Methylsulfanyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester

5-Methylsulfanyl-thiophene-2-carbaldehyde was annulated according toProcedure H (aldehyde and azido-acetic acid ethyl ester added as ethanolsolution (1 M of ester) to −20° C. ethoxide solution; allow to warm to10° C. over 4 h; reaction poured into cold saturated aqueous NH₄Cl;after ether extraction, acrylate organic phase washed with water untilaqueous phase was neutral; crude acrylate solution heated at reflux for2 h, allowed to cool to room temperature, stirred overnight).

CIMS m/e 240.0 ((M−H)⁺), 242.0 (MH⁺).

¹H NMR (CDCl₃) δ 9.05 (br s, 1H), 7.04 (m, 1H), 6.97 (s, 1H), 4.35 (q,J=7.1 Hz, 2H), 2.53 (s, 3H), 1.37 (t, J=7.1 Hz, 3H).

Example 48b

2-Methylsulfanyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid

2-Methylsulfanyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl esterwas hydrolyzed according to Procedure F (40° C. overnight).

CIMS m/e 211.9 ((M−H)⁺).

¹H NMR (DMSO-d₆)) δ 12.56 (s, 1H), 11.90 (s, 1H), 6.97 (s, 1H), 6.94 (s,1H), 2.47 (s, 1H).

The following compounds can also be prepared as set forth above:

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(3-hydroxy-azetidin-1-yl)-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(1,1-dioxo-1-thiazolidin-3-yl)-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-morpholin-4-yl-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3S,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-((3R,4R)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;and

2-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid[(1S)-benzyl-2-(4-hydroxy-piperidin-1-yl)-2-oxo-ethyl]-amide.

Biological Protocols

The utility of the compounds of the present invention as medical agentsin the treatment or prevention of diseases (such as are detailed herein)in animals, particularly mammals (e.g. humans) is demonstrated by theactivity of the compounds of this invention in conventional assays andthe in vitro and in vivo assays described below. Such assays alsoprovide a means whereby the activities of the compounds of thisinvention can be compared with the activities of other known compounds.The results of-these comparisons are useful for determining dosagelevels in animals, particularly mammals, including humans, for thetreatment of such diseases.

Glycogen Phosphorylase Production and Assays

The three different purified glycogen phosphorylase (GP) isoenzymes,wherein glycogen phosphorylase is in the activated “a” state (referredto as glycogen phosphorylase a, or the abbreviation GPa), and referredto here as human liver glycogen phosphorylase a (HLGPa), human muscleglycogen phosphorylase a (HMGPa), and human brain glycogen phosphorylasea (HBGPa), can be obtained by the following procedures.

Expression and Fermentation

The HLGP cDNAs (obtained as described in Newgard et al., Proc. Natl.Acad. Sci., 83: 8132-8136 (1986), and Newgard et al., Proc. Natl. Acad.Sci., 263: 3850-3857 (1988), respectively) and HMGP cDNAs (obtained byscreening a Stratagene (Stratagene Cloning Systems, La Jolla, Calif.)human muscle cDNA library with a polymerase chain reaction(PCR)-generated cDNA fragment based on information and methodologyreported for isolation of the human skeletal muscle glycogenphosphorylase gene and partial cDNA sequence by Kubisch et al., Centerfor Molecular Neurobiology, University of Hamburg, Martinistrasse 85,Hamburg, 20246 Germany; Genbank (National Center for BiotechnologyInformation, National Institutes of Health, USA) Accession NumbersU94774, U94775, U94776 and U94777, submitted Mar. 20, 1997; Burke etal., Proteins, 2:177-187 (1987); and Hwang et al., Eur. J. Biochem.,152: 267-274 (1985)) are expressed from plasmid pKK233-2 (PharmaciaBiotech. Inc., Piscataway, N.J.) in E. coli strain XL-1 Blue (StratageneCloning Systems, LaJolla, Calif.). The strain is inoculated into LBmedium (consisting of 10 g tryptone, 5 g yeast extract, 5 g NaCl, and 1ml 1 N NaOH per liter) plus 100 mg/L ampicillin, 100 mg/l pyridoxine and600 mg/L MnCl₂ and grown at 37° C. to a cell density of OD₅₅₀=1.0. Atthis point, the cells are induced with 1 mMisopropyl-1-thio-β-D-galactoside (IPTG). Three hours after induction thecells are harvested by centrifugation and cell pellets are frozen at−70° C. until needed for purification.

The HBGP cDNA can be expressed by several methodologies, for example, bythe method described by Crerar, et al. (J. Biol. Chem. 270:13748-13756(1995)). The method described by Crerar, et al. (J. Biol. Chem.,270:13748-13756 (1995)) for the expression of HBGP is as follows: theHBGP cDNA can be expressed from plasmid pTACTAC in E. coli strain 25A6.The strain is inoculated into LB medium (consisting of 10 g tryptone, 5g yeast extract, 5 g NaCl, and 1 ml 1N NaOH per liter) plus 50 mg/Lampicillin and grown overnight, then resuspended in fresh LB medium plus50 mg/L ampicillin, and reinoculated into a 40×volume of LB/amp mediacontaining 250 μM isopropyl-1-thio-β-D-galactoside (IPTG), 0.5 mMpyridoxine and 3 mM MnCl₂ and grown at 22° C. for 48-50 hours. The cellscan then be harvested by centrifugation and cell pellets are frozen at−70° C. until needed for purification.

The HLGP cDNA is expressed from plasmid pBlueBac III (Invitrogen Corp.,San Diego, Calif.) which is cotransfected with BaculoGold Linear ViralDNA (Pharmingen, San Diego, Calif.) into Sf9 Cells. Recombinant virus issubsequently plaque-purified. For production of protein, Sf9 Cells grownin serum-free medium (Sf-900 II serum free medium, Gibco BRL, LifeTechnologies, Grand Island, N.Y.) are infected at an moi of 0.5 and at acell density of 2×10⁶ Cells/ml. After growth for 72 hours at 27° C.,cells are centrifuged, and the cell pellets frozen at −70° C. untilneeded for purification.

Purification of Glycogen Phosphorylase Expressed in E. coli

The E. coli Cells in pellets described above are resuspended in 25 mMβ-glycerophosphate (pH 7.0) with 0.2 mM DTT, 1 mM MgCl₂, plus thefollowing protease inhibitors:

0.7 μg/ml Pepstatin A 0.5 μg/ml Leupeptin 0.2 mM phenylmethylsulfonylfluoride (PMSF), and 0.5 mM EDTA,

lysed by pretreatment with 200 μg/ml lysozyme and 3 μg/ml DNAasefollowed by sonication in 250 ml batches for 5×1.5 minutes on ice usinga Branson Model 450 ultrasonic cell disrupter (Branson Sonic Power Co.,Danbury Conn.). The E. coli Cell lysates are then cleared bycentrifugation at 35,000×g for one hour followed by filtration through0.45 micron filters. GP in the soluble fraction of the lysates(estimated to be less than 1% of the total protein) is purified bymonitoring the enzyme activity (as described in GPa Activity Assaysection, below) from a series of chromatographic steps detailed below.

Immobilized Metal Affinity Chromatography (IMAC)

This step is based on the method of Luong et al (Luong et al. Journal ofChromatography 584: 77-84 (1992)). Five hundred ml of the filteredsoluble fraction of cell lysates (prepared from approximately 160-250 gof original cell pellet) are loaded onto a 130 ml column of IMACChelating-Sepharose (Pharmacia LKB Biotechnology, Piscataway, N.J.)which has been charged with 50 mM CuCl₂ and 25 mM β-glycerophosphate,250 mM NaCl and 1 mM imidazole at pH 7 (equilibration buffer). Thecolumn is washed with equilibration buffer until the A₂₈₀ returns tobaseline. The sample is then eluted from the column with the same buffercontaining 100 mM imidazole to remove the bound GP and other boundproteins. Fractions containing the GP activity are pooled (approximately600 ml), and ethylenediaminetetraacetic acid (EDTA), DL-dithiothreitol(DTT), phenylmethylsulfonyl fluoride (PMSF), leupeptin and pepstatin Aare added to obtain 0.3 mM, 0.2 mM, 0.2 mM, 0.5 μg/ml and 0.7 μg/mlconcentrations respectively. The pooled GP is desalted over a SephadexG-25 Column (Sigma Chemical Co., St. Louis, Mo.) equilibrated with 25 mMTris-HCl (pH 7.3), 3 mM DTT buffer (Buffer A) to remove imidazole and isstored on ice and subjected to a second chromatographic step (below) ifnecessary.

5′-AMP-Sepharose Chromatography

The desalted pooled GP sample (approximately 600 mL) is next mixed with70 ml of 5′-AMP Sepharose (Pharmacia LKB Biotechnology, Piscataway,N.J.) which has been equilibrated with Buffer A (see above). The mixtureis gently agitated for one hour at 22° C. then packed into a column andwashed with Buffer A until the A₂₈₀ returns to baseline. GP and otherproteins are eluted from the column with 25 mM Tris-HCl, 0.2 mM DTT and10 mM adenosine 5′-monophosphate (AMP) at pH 7.3 (Buffer B).GP-containing fractions are pooled following identification bydetermining enzyme activity described below and visualizing the M,approximately 97 kdal GP protein band by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by silverstaining (2D-silver Stain II “Daiichi Kit”, Daiichi Pure Chemicals Co.,LTD., Tokyo, Japan) and then pooled. The pooled GP is dialyzed into 25mM β-glycerophosphate, 0.2 mM DTT, 0.3 mM EDTA, 200 mM NaCl, pH 7.0buffer (Buffer C) and stored on ice until use.

Prior to use of the GP enzyme, the enzyme is converted from the inactiveform as expressed in E. coli strain XL-1 Blue (designated GPb) (StrageneCloning Systems, La Jolla, Calif.), to the active form (designated GPa)by the procedure described in Section (A) Activation of GP below.

Purification of Glycogen Phosphorylase Expressed in Sf9 Cells

The Sf9 Cells in pellets described above are resuspended in 25 mMβ-glycerophosphate (pH 7.0) with 0.2 mM DTT, 1 mM MgCl2, plus thefollowing protease inhibitors:

0.7 μg/ml Pepstatin A 0.5 μg/ml Leupeptin 0.2 mM phenylmethylsulfonylfluoride (PMSF), and 0.5 mM EDTA,

lysed by pretreatment with 3 μg/ml DNAase followed by sonication inbatches for 3×1 minutes on ice using a Branson Model 450 ultrasonic celldisrupter (Branson Sonic Power Co., Danbury Conn.). The Sf9 Cell lysatesare then cleared by centrifugation at 35,000×g for one hour followed byfiltration through 0.45 micron filters. GP in the soluble fraction ofthe lysates (estimated to be 1.5% of the total protein) is purified bymonitoring the enzyme activity (as described in GPa Activity Assaysection, below) from a series of chromatographic steps detailed below.

Immobilized Metal Affinity Chromatography (IMAC)

Immobilized Metal Affinity Chromatography is performed as described inthe section above. The pooled, desalted GP is then stored on ice untilfurther processed.

Activation of GP

Before further chromatography, the fraction of inactive enzyme asexpressed in Sf9 Cells (designated GPb) is converted to the active form(designated GPa) by the following procedure described in Section (A)Activation of GP below.

Anion Exchange Chromatography

Following activation of the IMAC purified GPb to GPa by reaction withthe immobilized phosphorylase kinase, as described below, the pooled GPafractions are dialyzed against 25 mM Tris-HCl, pH 7.5, containing 0.5 mMDTT, 0.2 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1.0 μg/mlleupeptin and 1.0 μg/ml pepstatin A. The fraction is then loaded onto aMonoQ Anion Exchange Chromatography column (Pharmacia Biotech. Inc.,Piscataway, N.J.). The column is washed with equilibration buffer untilthe A₂₈₀ returns to baseline. The sample is then eluted from the columnwith a linear gradient of 0-0.25 M NaCl to remove the bound GP and otherbound proteins. GP-containing fractions elute between 0.1-0.2 M NaClrange, as detected by monitoring the eluant for peak protein absorbanceat A₂₈₀. The GP protein is then identified by visualizing the M_(r)approximately 97 kdal GP protein band by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by silverstaining (2D-silver Stain II “Daiichi Kit”, Daiichi Pure Chemicals Co.,LTD., Tokyo, Japan) and then pooled. The pooled GP is dialyzed into 25mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 1.0 mMDTT, 0.5 mM EDTA, 5 mM NaCl, pH 6.8 buffer and stored on ice until use.

Determination of GP Enzyme Activity

A) Activation of GP: Conversion of GPb to GPa

Prior to the determination of GP enzyme activity, the enzyme isconverted from the inactive form as expressed in E. coli strain XL-1Blue (designated GPb) (Stragene Cloning Systems, La Jolla, Calif.), tothe active form (designated GPa) by phosphorylation of GP usingphosphorylase kinase as follows. The fraction of inactive enzyme asexpressed in Sf9 Cells (designated GPb) is also converted to the activeform (designated GPa) by the follow procedure.

GP Reaction with Immobilized Phosphorylase Kinase

Phosphorylase kinase (Sigma Chemical Company, St. Louis, Mo.) isimmobilized on Affi-Gel® 10 (BioRad Corp., Melvile, N.Y.) as per themanufacturer's instructions. In brief, the phosphorylase kinase enzyme(10 mg) is incubated with washed Affi-Gel® beads (1 ml) in 2.5 ml of 100mM HEPES and 80 mM CaCl₂ at pH 7.4 for 4 hours at 4° C. The Affi-Gel®beads are then washed once with the same buffer prior to blocking with50 mM HEPES and 1 M glycine methyl ester at pH 8.0 for one hour at roomtemperature. Blocking buffer is removed and replaced with 50 mM HEPES(pH 7.4), 1 mM β-mercaptoethanol and 0.2% NaN₃ for storage. Prior to useto convert GPb to GPa, the Affi-Gel® immobilized phosphorylase kinasebeads are equilibrated by washing in the buffer used to perform thekinase reaction, consisting of 25 mM β-glycerophosphate, 0.3 mM DTT, and0.3 mM EDTA at pH 7.8 (kinase assay buffer).

The partially purified, inactive GPb obtained from 5′-AMP-Sepharosechromatography above (from E. coli) or the mixture of GPa and GPbobtained from IMAC above (from Sf9 Cells) is diluted 1:10 with thekinase assay buffer then mixed with the aforementioned phosphorylasekinase enzyme immobilized on the Affi-Gel® beads. NaATP is added to 5 mMand MgCl₂ to 6 mM. The resulting mixture is mixed gently at 25° C. for30 to 60 minutes. The activated sample is removed from the beads and thepercent activation of GPb by conversion to GPa is estimated bydetermining GP enzyme activity in the presence and absence of 3.3 mMAMP. The percentage of total GP enzyme activity due to GPa enzymeactivity (AMP-independent) is then calculated as follows:${\% \quad {of}\quad {total}\quad {HLGPa}} = \frac{{{HLGP}\quad {activity}} - {AMP}}{{{HLGP}\quad {activity}} + {AMP}}$

Alternately, the conversion of GPb to GPa can be monitored byisoelectric focusing, based on the shift in electrophoretic mobilitythat is noted following conversion of GPb to GPa. GP samples areanalyzed by isoelectric focusing (IEF) utilizing the Pharmacia PfastGelSystem (Pharmacia Biotech. Inc., Piscataway, N.J.) using precast gels(pl range 4-6.5) and the manufacturer's recommended method. The resolvedGPa and GPb bands are then visualized on the gels by silver staining(2D-silver Stain II “Daiichi Kit”, Daiichi Pure Chemicals Co., LTD.,Tokyo, Japan). Identification of GPa and GPb is made by comparison to E.coli derived GPa and GPb standards that are run in parallel on the samegels as the experimental samples.

B) GPa Activity Assay

The disease/condition treating/preventing activities described herein ofthe compounds of the present invention can be indirectly determined byassessing the effect of the compounds of this invention on the activityof the activated form of glycogen phosphorylase (GPa) by one of twomethods; glycogen phosphorylase a activity is measured in the forwarddirection by monitoring the production of glucose-1-phosphate fromglycogen or by following the reverse reaction, measuring glycogensynthesis from glucose-1-phosphate by the release of inorganicphosphate. All reactions are run in triplicate in 96-well microtiterplates and the change in absorbance due to formation of the reactionproduct is measured at the wavelength specified below in a MCC/340 MKIIElisa Reader (Lab Systems, Finland), connected to a Titertech MicroplateStacker (ICN Biomedical Co, Huntsville, Ala.).

To measure the GPa enzyme activity in the forward direction, theproduction of glucose-1-phosphate from glycogen is monitored by themultienzyme coupled general method of Pesce et al. [Pesce, M. A.,Bodourian, S. H., Harris, R. C. and Nicholson, J. F. Clinical Chemistry23: 1711-1717 (1977)] modified as follows: 1 to 100 μg GPa, 10 unitsphosphoglucomutase and 15 units glucose-6-phosphate dehydrogenase(Boehringer Mannheim Biochemicals, Indianapolis, Ind.) are diluted to 1mL in Buffer D (pH 7.2, 50 mM HEPES, 100 mM KCl, 2.5 mMethyleneglycoltetraacetic acid (EGTA), 2.5 mM MgCl₂, 3.5 mM KH₂PO₄ and0.5 mM dithiothreitol). Twenty μl of this stock is added to 80 μl ofBuffer D containing 0.47 mg/mL glycogen, 9.4 mM glucose, 0.63 mM of theoxidized form of nicotinamide adenine dinucleotide phosphate (NADP+).The compound to be tested is added as 5 μl of solution in 14%dimethylsulfoxide (DMSO) prior to the addition of the enzymes. The basalrate of GPa enzyme activity in the absence of inhibitors, e.g., acompound of this invention, is determined by adding 5 μl of 14% DMSO anda fully-inhibited rate of GPa enzyme activity is obtained by adding 20μl of 50 mM of the positive control test substance, caffeine. Thereaction is followed at room temperature by measuring the conversion ofoxidized NADP+ to reduced NADPH at 340 nm.

To measure the GPa enzyme activity in the reverse direction, theconversion of glucose-1-phosphate into glycogen plus inorganic phosphateis measured by the general method described by Engers et al. [Engers, H.D., Shechosky, S. and Madsen, N.B., Can. J. Biochem. 48: 746-754 (1970)]modified as follows: 1 to 100 μg GPa is diluted to 1 ml in Buffer E (pH7.2, 50 mM HEPES, 100 mM KCl, 2.5 mM EGTA, 2.5 mM MgCl₂ and 0.5 mMdithiothreitol). Twenty μl of this stock is added to 80 μl of Buffer Ewith 1.25 mg/ml glycogen, 9.4 mM glucose, and 0.63 mMglucose-1-phosphate. The compound to be tested is added as 5 μl ofsolution in 14% DMSO prior to the addition of the enzyme. The basal rateof GPa enzyme activity in the absence of added inhibitors, e.g., acompound of this invention, is determined by adding 5 μl of 14% DMSO anda fully-inhibited rate of GPa enzyme activity is obtained by adding 20μL of 50 mM caffeine. This mixture is incubated at room temperature for1 hour and the inorganic phosphate released from the glucose-1-phosphateis measured by the general method of Lanzetta et al. [Lanzetta, P.A.,Alvarez, L. J., Reinach, P. S. and Candia, O. A. Anal. Biochem. 100:95-97 (1979)] modified as follows: 150 μl of 10 mg/ml ammoniummolybdate, 0.38 mg/ml malachite green in 1 N HCl is added to 100 μl ofthe enzyme mix. After a 20 minute incubation at room temperature, theabsorbance is measured at 620 nm.

The above assays carried out with a range of concentrations of testcompound allows the determination of an IC₅₀ value (concentration oftest compound required for 50% inhibition) for the in vitro inhibitionof GPa enzyme activity by that test compound.

The compounds of this invention are readily adapted to clinical use ashypoglycemic agents. The hypoglycemic activity of the compounds of thisinvention can be determined by the amount of test compound that reducesglucose levels relative to a vehicle without test compound in male ob/obmice. The test also allows the determination of an approximate minimaleffective dose (MED) value for the in vivo reduction of plasma glucoseconcentration in such mice for such test compounds.

Since the concentration of glucose in blood is closely related to thedevelopment of diabetic disorders, the compounds of the presentinvention, by virtue of their hypoglycemic action, prevent, arrestand/or regress diabetic disorders.

Five to eight week old male C57BL/6J-ob/ob mice (obtained from JacksonLaboratory, Bar Harbor, Me.) are housed five per cage under standardanimal care practices. After a one week acclimation period, the animalsare weighed and 25 microliters of blood are collected from theretro-orbital sinus prior to any treatment. The blood sample isimmediately diluted 1:5 with saline containing 0.025% sodium heparin,and held on ice for metabolite analysis. Animals are assigned totreatment groups so that each group has a similar mean for plasmaglucose concentration. After group assignment, animals are dosed orallyeach day for four days with the vehicle consisting of either: (1) 0.25%w/v methyl cellulose in water without pH adjustment; or (2) 0.1%Pluronice P105 Block Copolymer Surfactant (BASF Corporation, Parsippany,N.J.) in 0.1% saline without pH adjustment. On day 5, the animals areweighed again and then dosed orally with a test compound or the vehiclealone. All compounds are administered in vehicle consisting of either:(1) 0.25% w/v methyl cellulose in water; or 3) neat PEG 400 without pHadjustment; (2) 10% DMSO/0.1% Pluronice in 0.1% saline without pHadjustment; or 3) neat PEG 400 without pH adjustment. The animals arethen bled from the retro-orbital sinus three hours later fordetermination of blood metabolite levels. The freshly collected samplesare centrifuged for two minutes at 10,000×g at room temperature. Thesupernatant is analyzed for glucose, for example, by the Abbott VP™(Abbott Laboratories, Diagnostics Division, Irving, Tex.) and VP SuperSystem® Autoanalyzer (Abbott Laboratories, Irving, Tex.), or by theAbbott Spectrum CCX™ (Abbott Laboratories, Irving, Tex.) using theA-Gent™Glucose-UV Test reagent system (Abbott Laboratories, Irving,Tex.) (a modification of the method of Richterich and Dauwalder,Schweizerische Medizinische Wochenschrift, 101: 860 (1971)) (hexokinasemethod) using a 100 mg/dl standard. Plasma glucose is then calculated bythe equation:

Plasma glucose(mg/dl)=Sample value×8.14

where 8.14 is the dilution factor, adjusted for plasma hematocrit(assuming the hematocrit is 44%).

The animals dosed with vehicle maintain substantially unchangedhyperglycemic glucose levels (e.g., greater than or equal to 250 mg/dl),animals treated with compounds having hypoglycemic activity at suitabledoses have significantly depressed glucose levels. Hypoglycemic activityof the test compounds is determined by statistical analysis (unpairedt-test) of the mean plasma glucose concentration between the testcompound group and vehicle-treated group on day 5. The above assaycarried out with a range of doses of a test compound allows thedetermination of an approximate minimal effective dose (MED) value forthe in vivo reduction of plasma glucose concentration.

The compounds of the present invention are readily adapted to clinicaluse as hyperinsulinemia reversing agents, triglyceride lowering agentsand hypocholesterolemic agents. Such activity can be determined by theamount of test compound that reduces insulin, triglycerides orcholesterol levels relative to a control vehicle without test compoundin male ob/ob mice.

Since the concentration of cholesterol in blood is closely related tothe development of cardiovascular, cerebral vascular or peripheralvascular disorders, the compounds of this invention, by virtue of theirhypocholesterolemic action, prevent, arrest and/or regressatherosclerosis.

Since the concentration of insulin in blood is related to the promotionof vascular cell growth and increased renal sodium retention, (inaddition to the other actions, e.g., promotion of glucose utilization)and these functions are known causes of hypertension, the compounds ofthis invention, by virtue of their hypoinsulinemic action, prevent,arrest and/or regress hypertension.

Since the concentration of triglycerides in blood contributes to theoverall levels of blood lipids, the compounds of this invention, byvirtue of their triglyceride lowering and/or free fatty acid loweringactivity prevent, arrest and/or regress hyperlipidemia.

Free fatty acids contribute to the overall level of blood lipids andindependently have been negatively correlated with insulin sensitivityin a variety of physiologic and pathologic states.

Five to eight week old male C57BL/6J-ob/ob mice (obtained from JacksonLaboratory, Bar Harbor, Me.) are housed five per cage under standardanimal care practices and fed standard rodent diet ad libitum. After aone week acclimation period, the animals are weighed and 25 microlitersof blood are collected from the retro-orbital sinus prior to anytreatment. The blood sample is immediately diluted 1:5 with salinecontaining 0.025% sodium heparin, and held on ice for plasma glucoseanalysis. Animals are assigned to treatment groups so that each grouphas a similar mean for plasma glucose concentration. The compound to betested is administered by oral gavage as an about 0.02% to 2.0% solution(weight/volume (w/v)) in either (1) 10% DMSO/0.1% Pluronic® P105 BlockCopolymer Surfactant (BASF Corporation, Parsippany, N.J.) in 0.1% salinewithout pH adjustment or (2) 0.25% w/v methylcellulose in water withoutpH adjustment. Alternatively, the compound to be tested can beadminsitrered by oral gavage dissolved in or in suspension in neat PEG400. Single daily dosing (s.i.d.) or twice daily dosing (b.i.d.) ismaintained for 1 to, for example, 15 days. Control mice receive the 10%DMSO/0.1% Pluronic® P105 in 0.1% saline without pH adjustment or the0.25% w/v methylcellulose in water without pH adjustment, or the neatPEG 400 without pH adjustment.

Three hours after the last dose is administered, the animals aresacrificed by decapitation and trunk blood is collected into 0.5 mlserum separator tubes containing 3.6 mg of a 1:1 weight/weight sodiumfluoride: potassium oxalate mixture. The freshly collected samples arecentrifuged for two minutes at 10,000×g at room temperature, and theserum supernatant is transferred and diluted 1:1 volume/volume with a 1TIU/ml aprotinin solution in 0.1% saline without pH adjustment.

The diluted serum samples are then stored at −80° C. until analysis. Thethawed, diluted serum samples are analyzed for insulin, triglycerides,free fatty acids and cholesterol levels. Serum insulin concentration isdetermined using Equate® RIA INSULIN kits (double antibody method; asspecified by the manufacturer) available from Binax, South Portland, Me.The inter assay coefficient of variation is ≦10%. Serum triglyceridesare determined using the Abbott VP™ and VP Super System® Autoanalyzer(Abbott Laboratories, Irving, Tex.), or the Abbott Spectrum CCX™ (AbbottLaboratories, Irving, Tex.) using the A-Gent™ Triglycerides Test reagentsystem (Abbott Laboratories, Diagnostics Division, Irving, Tex.)(lipase-coupled enzyme method; a modification of the method of Sampson,et al., Clinical Chemistry 21: 1983 (1975)). Serum total cholesterollevels are determined using the Abbott VP™ and VP Super System®Autoanalyzer (Abbott Laboratories, Irving, Tex.), and A-Gent™Cholesterol Test reagent system (cholesterol esterase-coupled enzymemethod; a modification of the method of Allain, et al. ClinicalChemistry 20: 470 (1974)) using 100 and 300 mg/dl standards. Serum freefatty acid concentration is determined utilizing a kit from AmanoInternational Enzyme Co., Inc., as adapted for use with the Abbott VP™and VP Super System® Autoanalyzer (Abbott Laboratories, Irving, Tex.),or the Abbott Spectrum CCX™ (Abbott Laboratories, Irving, Tex.). Seruminsulin, triglycerides, free fatty acids and total cholesterol levelsare then calculated by the equations,

Serum insulin(μU/ml)=Sample value×2

Serum triglycerides(mg/dl)=Sample value×2

Serum total cholesterol(mg/dl)=Sample value×2

Serum free fatty acid (μEq/l)=Sample value×2

where 2 is the dilution factor.

The animals dosed with vehicle maintain substantially unchanged,elevated serum insulin (e.g., 275 μU/ml), serum triglycerides (e.g., 235mg/dl), serum free fatty acid (1500 mEq/ml) and serum total cholesterol(e.g., 190 mg/dl) levels, while animals treated with compounds of thepresent invention generally display reduced serum insulin,triglycerides, free fatty acid and total cholesterol levels. The seruminsulin, triglycerides, free fatty acid and total cholesterol loweringactivity of the test compounds are determined by statistical analysis(unpaired t-test) of the mean serum insulin, triglycerides, or totalcholesterol concentration between the test compound group and thevehicle-treated control group.

What is claimed is:
 1. A compound of formula I:

a stereoisomer, pharmaceutically acceptable salt or prodrug thereof, ora pharmaceutically acceptable salt of the prodrug, wherein Q is aryl,substituted aryl, heteroaryl, or substituted heteroaryl; each Z and Xare independently (C, CH or CH₂), N, O or S; X¹ is NR^(a), —CH₂—, O orS; each - - - - is independently a bond or is absent, provided thatboth - - - - are not simultaneously bonds; R¹ is hydrogen, halogen,—OC₁-C₈alkyl, —SC₁-C₈alkyl, —C₁-C₈alkyl, —CF₃, —NH₂, —NHC₁-C₈alkyl,—N(C₁-C₈alkyl)₂, —NO₂, —CN, —CO₂H, —CO₂C₁-C₈alkyl, —C₂-C₈alkenyl, or—C₂-C₈akynyl; each R^(a) and R^(b) is independently hydrogen or—C₁-C₈alkyl; Y is

 or absert; R² and R³ together with the atoms on the ring to which theyare attached form a five or six membered ring consisting of from 0 to 3heteroatoms and from 0 to 2 double bonds; R⁴ is —C(═O)-A; A is—NR^(d)R^(d), —NR^(a)CH₂CH₂OR^(a),

each R^(d) is independently hydrogen, C₁-C₈alkyl, C₁-C₈alkoxy, aryl,substituted aryl, heteroaryl, or substituted heteroaryl; each R^(c) isindependently hydrogen, —C(═O)OR^(a), —OR^(a), —SR^(a), or —NR^(a)R^(a);and each n is independently 1-3.
 2. A compound of claim 1 wherein R^(b)and R¹ are hydrogen.
 3. A compound of claim 1 wherein R^(b) is hydrogen;R¹ is hydrogen; Y is

and A is


4. A compound of claim 1 wherein R^(b) is hydrogen; R¹ is hydrogen; Y isabsent; and A is


5. A compound of claim 1 wherein R^(b) is hydrogen; R¹ is hydrogen; Z isC; X is O or S; Y is absent; and A is


6. A compound of claim 1 wherein Q is phenyl and A is


7. A pharmaceutical composition comprising a compound of claim 1, astereoisomer, pharmaceutically acceptable salt or prodrug thereof, or apharmaceutically acceptable salt of the prodrug.
 8. A method of treatingdiabetes the method comprising the step of administering to a patienthaving diabetes a therapeutically effective amount of a compound ofclaim 1, a stereoisomer, pharmaceutically acceptable salt or prodrugthereof, or a pharmaceutically acceptable salt of the prodrug.
 9. Acompound selected from the group consisting of:[4H-1,7-dithia-4-aza-cyclopenta [a]pentalene-5-carboxylic acid[(1S)-benzyl-3-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-(2R)-hydroxy-3-oxo-propyl]-amide;and 4H-1,7-dithia-4-aza-cyclopenta [a]pentalene-5-carboxylic acid[(1S)-benzyl-2-((3R,4S)-dihydroxy-pyrrolidin-1-yl)-2-oxo-ethyl]-amide;or a stereoisomer, pharmaceutically acceptable salt or prodrug of thecompound, or a pharmaceutically acceptable salt of the prodrug.